Tumour virus deregulation of the cell cycle: translational control and function of RGC-32

We have been studying a human protein (RGC-32) that may be involved in the development of many cancers. We found that RGC-32 is produced when white blood cells are infected by the cancer virus Epstein-Barr virus, suggesting that RGC-32 may be involved in Epstein-Barr virus-associated cancers. These include many lymphomas e.g. Burkitt's, Hodgkin's and post-transplant lymphoma. We have shown that if we reduce RGC-32 production in Epstein-Barr virus infected white blood cells, they stop growing. This tells us that inducing the production of RGC-32 may be an important part of how Epstein-Barr virus drives white blood cells to keep growing indefinitely and become cancer cells. We have discovered news ways in which RGC-32 production is controlled in white blood cells.

Our research will obtain further information on how RGC-32 production is controlled by Epstein-Barr virus and uncover how RGC-32 controls the growth of cells. This research will therefore increase our understanding of how production of RGC-32 may contribute to cancer development. In the longer term our work could open up possibilities for designing drugs to block the production or function of RGC-32 that could help in treatment of Epstein-Barr virus associated cancers and some other cancers where RGC-32 is produced at high level (e.g. breast and colon).

Specifically we will determine:

1. How Epstein-Barr virus drives the production of RGC-32 in white blood cells.
2. How RGC-32 controls the growth of cells by affecting the different stages of the cell growth cycle.
3. How RGC-32 associates with growth control proteins and how this affects how they function.

Our research will provide important information for both basic scientists and clinicians working in the field of cancer research. It could inform longer-term therapeutic strategies that may eventually benefit patients.

The disruption of cell-cycle control by Epstein-Barr virus (EBV) plays an important role in growth transformation, but the mechanisms involved are not fully defined. RGC-32 is translationally upregulated on B-cell infection by EBV and is deregulated in numerous cancers. RGC-32 may influence cell growth by regulating the mitotic kinases, CDK1 and PLK1, but the molecular details are unknown. We have shown that RGC-32 is essential for the growth of EBV-transformed B cells and that the translational repressor, Pumilio, can control its translation.

To decipher the role of RGC-32 in viral and non-viral oncogenesis we will study its regulation, function and structure. We will:

1. Investigate the role of specific RNA-binding proteins and stress granules (SGs) in RGC-32 translation control in EBV-infected cells.
We will study this in EBV-infected B cell lines and on primary B-cell infection using immunofluorescence, RNA-FISH, Western blotting and RNAi.
2. Investigate whether EBV latent proteins are responsible for activating RGC-32 translation.
We will interfere with the function of EBV latent proteins and pathways activated by them and use knock-out cell-lines to determine the influence of specific EBV proteins on the activity or expression of the proteins identified in (1) that control RGC-32 translation.
3. Determine the role of RGC-32 in the regulation of mitosis and response to DNA damage.
Using chemical genetics in tractable epithelial cell lines in which we have shown that RGC-32 promotes mitotic entry, we will examine the influence of RGC-32 on mitotic phenotypes and cell-cycle checkpoints through effects on CDK1 and PLK1.
4. Obtain molecular information on the structure and function of RGC-32 complexes.
With recombinant proteins, we will determine the mode, sites of association and effects of RGC-32 binding to CDK1-cyclin B1 and PLK1 using FRET-based binding assays and peptide substrate screening. We will also obtain 3D structures of RGC-32 complexes.

Who will benefit?
This research will provide direct benefit to the researchers employed on this grant in the form of training in new techniques and methods.The discipline-spanning nature of this research proposal on RGC-32 means that the beneficiaries of this research will include scientists across a wide number of fields both within the UK and internationally. These include researchers interested in mechanisms of viral transformation, cancer biology and basic translational control mechanisms. Our findings could also benefit clinicians treating EBV-associated tumours and other cancers where RGC-32 deregulation has been implicated. In the longer term the information we obtain on the regulation and function of RGC-32 could inform the design of future therapeutic strategies to target RGC-32 in EBV-associated and non-viral tumours. This could be of interest to the pharmaceutical industry, especially those sectors involved in drug development and design.
How will they benefit?
By carrying out this research the post-doctoral fellows will expand their skill set and receive training (as required) in a range of biochemical and molecular biology techniques, microscopy, protein-RNA interaction assays, cell-cycle analysis and RNAi techniques. This research will also benefit these researchers by increasing their academic knowledge of cell-cycle and translational control mechanisms by engaging with the literature and attending international meetings. They will benefit from further communication skill training when delivering poster and oral presentations.
This research will also enhance the knowledge economy by providing important new scientific knowledge that will help scientific progress across a number of disciplines that are being actively investigated globally.