Understanding the cellular pathways regulated by Dis3L2 in cell proliferation.

Regulation of cell proliferation is of crucial importance to all multicellular organisms. Cells must proliferate throughout development in order for the organism to grow from an egg to an adult. Proliferation must also occur to repair damaged areas during the process of wound healing. Control of proliferation is vitally important to allow individual animals and their constituent organs to grow to reach and not exceed their correct sizes, as well as to maintain symmetry between the left and right sides of an animal. Uncontrolled cell proliferation is a hallmark of cancer with many genes involved in growth and proliferation implicated in cancer progression.

Using fruit flies as a model organism, we have recently discovered that cell proliferation can be regulated by an enzyme named Dis3L2. This enzyme is known to destroy messenger RNA molecules (mRNAs) which instruct the cell to make particular proteins. By comparing mutant fruit flies lacking Dis3L2 with normal individuals, we have found that lack of Dis3L2 results in wings that are much larger than normal. The wings grow from larval wing imaginal discs, which are also much larger in the mutant. Our results are particularly interesting because mutations in the equivalent human gene, DIS3L2, result in Perlman syndrome and susceptibility to a kidney cancer called Wilms' tumour. Perlman syndrome is an overgrowth condition where affected children display pre-natal gigantism and abnormal enlargement of organs (e.g. kidneys). Therefore mutations in Dis3L2 in both fruit flies and humans result in the overgrowth of cells within some organs, showing an excellent conservation of this biological pathway between these organisms and demonstrating the usefulness of fruit flies to understand this disease.

Using state-of-the-art molecular methods, we have discovered that a lack of Dis3L2 results in an increase in levels of a few specific mRNAs. The known function of some of these mRNAs suggest a molecular pathway for understanding the function of Dis3L2 in flies and humans. Our hypothesis is that Dis3L2 normally controls proliferation by limiting energy production within the cell as well as regulating the levels of important cellular resources. Since proliferating cells require more energy to fuel their rapid growth, this hypothesis is consistent with the tissue overgrowth we see in our fruit flies. We have the expertise, as well as the molecular and genetic tools to test this hypothesis.

The proposed project is entirely novel; as yet no research group has elucidated the mechanisms whereby Dis3L2 controls proliferation in the natural context of a developing organism. Previous studies have used individual tissue culture cells or immortalised cells in culture rather than normal cells in a natural tissue therefore have missed clues about the cellular pathways involved. Since the cellular pathways controlling growth in fruit flies are very similar to those in humans, the knowledge gained during this project may help us to to understand the ways that normal tissues grow and develop. This knowledge will also be useful in finding ways to promote controlled regeneration of tissues such as that which occurs during liver regeneration. It will also be important in the search for therapies to combat uncontrolled proliferation, as occurs during cancer. Should our experiments confirm that Dis3L2 controls proliferation via metabolic pathways, this will provide a powerful way of combatting cancer as it will be difficult for cancer cells to circumvent their need for high metabolic rates. This project will therefore provide valuable insights into a new cellular pathway which can be used in the development of new disease therapeutics.

Dis3L2 is a member of the highly conserved RNaseII/RNB family of 3'-5' exoribonucleases. Recent work has shown that Dis3L2 is primarily cytoplasmic and acts independently of the exosome to degrade both mRNAs and non-coding RNAs. Mutations in human DIS3L2 are associated with Perlman syndrome, which is a congenital overgrowth syndrome with Wilms' tumour susceptibility. The molecular pathways whereby Dis3L2 affects proliferation have not yet been determined because a model system which recapitulates this tissue proliferation has not been available.

Using Drosophila as a model system, we have shown that null mutations and knockdown of Dis3L2 result in larvae with substantially larger wing imaginal discs and adult wings compared to isogenic controls. This is due to increased cellular proliferation rather than an increase in cell size. Therefore, we have discovered an excellent experimental system to dissect the cellular mechanisms underlying the ways in which Dis3L2 controls proliferation in the context of a developing organism. Using RNA-seq we identified a small set of mRNAs that are sensitive to Dis3L2 activity including pyrexia (a TRP cation channel) and cyt-c-d (involved in mitochondrial respiration).

The overall aim of this project is to understand the molecular mechanisms whereby Dis3L2 controls proliferation. Our hypothesis is Dis3L2 regulates fundamental metabolic changes such as energy production and cation levels. Since proliferating cells including cancer cells require high metabolic activity to provide the building blocks for rapid growth, this hypothesis is consistent with the observed phenotypes. Using a range of genetic and biochemical techniques we will test this hypothesis and so gain understanding of the molecular mechanisms involved. Since Dis3L2 is highly conserved throughout evolution, it is likely that the mechanisms regulating proliferation via Dis3L2 will have parallels in other organisms.

Who will benefit from this research?
The main beneficiaries of the proposed work comprise those in the Pharmaceutical industry, Clinicians, Biomedical Scientists and the General Public (including schoolchildren). Although this project is primarily "basic, blue-sky research" it nevertheless will lead to new insights important for therapeutics in the future.

How will they benefit from this research?
1. Pharmaceutical Industry and Biotechnology
Since the proposed project aims to understand a new pathway controlling proliferation, it is highly likely that we will find new "druggable" targets for cancer and to promote regeneration (e.g. liver regeneration). We will therefore aim to collaborate with the Sussex Drug Discovery Centre to find molecules which affect the activity of Dis3L2 or downstream targets. The proposed research is also of relevance to industrialists as it may provide a pathway to molecular therapeutics based on the modulation of RNA stability. The project will also be highly relevant to our work on microRNA biomarkers. This is because it will shed light on the 3' end modification and degradation of disease-relevant microRNAs and non-coding RNAs as well as benefit from our expertise in methodologies (e.g RNA-seq). Our previous BBSRC-funded research led to work on microRNAs/non-coding RNAs as biomarkers in myeloma, sepsis, melanoma and Motor Neurone Disease (ALS) resulting in 2 publications (2 in prep) and 2 patents. We are therefore familiar with working with the Sussex Innovation Centre to market and patent microRNA biomarkers. We also have been awarded a BBSRC "Sparking Impact" Award to market and patent microRNA biomarkers in melanoma.

2. Clinicians
Clinicians will benefit from the proposed research because the project will provide fundamental insights which will be relevant to their research on cancer and microRNA biomarkers. With the work taking place in a Medical School there is ample scope to engage clinicians and medical researchers to take advantage of the knowledge and expertise we have developed. Indeed, clinicians have already benefitted from our previous BBSRC-funded research, particularly in the area of microRNA biology and cancer. We are currently collaborating with 3 clinical research groups to explore the use of microRNAs as biomarkers in a variety of human diseases. The proposed project will allow cross-fertilization of ideas on medically related topics which will therefore contribute to our continued efforts to enhance the quality of life in the UK.

3. General Public and Schools
We think it is important to disseminate our research to a wider public because we are interested in fundamental problems that are ultimately relevant to human health. The main theme of the work, concerning gene regulation of RNAs involved in proliferation, will be both surprising and fascinating to the public, particularly as there are clinical implications. We plan to present our work at the Brighton Science Festival, as well as at Open Days and to visiting schoolchildren.

4. Capacity building for doctoral and post-doctoral researchers.
The research skills gained by both the post-doc and the research technician will be valuable to their future careers and applicable to Biomedical Industries. These research skills include RNA-seq, management of large data sets and networking and communications skills. The usefulness of these skills is demonstrated by one of my previous post-docs who is now working in a spin-out company in Cambridge to devise ways to detect circulating nucleic acids. In addition, the Co-applicant Chris Jones, who now has a permanent position as a Medical Statistician at BSMS, gained his bioinformatic skills by working as a BBSRC-funded postdoc in my lab. The post-doc funded on the grant will be in a position to teach high-level techniques to PhD students including clinical undergraduate/PhD/MD students as well as NHS Biomedical Scientists.