IDENTIFICATION OF NUCLEAR PATHWAYS THAT ARE CAUSALLY INVOLVED IN NUCLEOLAR TARGETING OF NF-KAPPAB/RELA.

Colorectal cancer is the most common cause of cancer death amongst the non-smoking population in the UK and is a major public health issue for the British population. Unequivocal evidence indicates that aspirin and related agents can prevent colorectal cancer and cause regression of this disease. However, the potential of aspirin-like agents is limited by their toxicity. In this lab, we have been undertaking studies to understand how aspirin-like agents act against colon cancer cells, in order to develop more effective and specific alternatives. We have shown that these agents cause a molecule involved in the regulation of cell growth and death, RelA, to move from the cytoplasmic compartment of the cell to the nucleoplasmic compartment then to a nuclear compartment called the nucleolus. We have also shown that movement of RelA from the nucleoplasm to the nucleolus is important for the ability of aspirin-like agents to kill colon cancer cells. Furthermore, we have demonstrated that artificially localising RelA to the nucleolus mediates the death of this cell type
The overall objective of this study is to understand how aspirin causes RelA to move from the nucleoplasm to the nucleolus. Identifying the cellular pathways involved will allow the design of preventative/therapeutic agents that force RelA into the nucleolar compartment to kill colon cancer cells. We have already shown that a molecule called COMMD1 is important in causing RelA to go to the nucleolus in response to aspirin and that COMMD1 acts by linking a regulatory molecule called ubiquitin, to RelA.

The specific objectives of this proposal are:

1. To identify molecules that, alongside COMMD1, are important for linking ubiquitin to RelA after aspirin treatment. We will examine the role of candidate molecules that have previously been identified as playing a role in linking ubiquitin to RelA. We will also isolate COMMD1 from aspirin treated cells and use a technique called mass spectrometry to identify molecules that bind to COMMD1 in response to the agent. We will examine how aspirin effects these molecules and how aspirin upregulates COMMD1.
2. Identification of the specific parts of RelA that are linked to ubiquitin in response to aspirin and the nature of this linkage. Ubiquitin is generally linked onto proteins at specific sites called lysines. We will identify the lysines of RelA that ubiquitin is linked to in response to aspirin. Ubiquitin also links to itself on lysine molecules to form chains. We will determine which lysine on ubiquitin is critical for nucleolar translocation of RelA using, amongst other approaches, a single cell assay where ubiquitination can be visualised.
3. Identification of proteins that transport ubiquitin-linked RelA to the nucleolus. When ubiquitin is linked to a molecule, specific proteins bind to that molecule. Therefore, we suggest that when ubiquitin is linked to RelA, specific molecules bind RelA and transport the protein to the nucleolus. We will use a labelling approach and mass spectrometry to identify proteins that bind specifically to aspirin-induced, ubiquitin-linked RelA.

These complementary approaches will allow us to understand the basic science of how RelA is regulated in the nucleoplasm, how linking ubiquitin to RelA causes it to go to the nucleolus and how proteins similar to RelA locate to different compartments in the cell. More importantly, these studies may reveal a way to chemically manipulate these pathways to mimic effects of aspirin on colon cancer cells.

Objective 1. Identification of the COMMD1 complex that ubiquitinates RelA in response to aspirin. Approaches that will be used to complete this objective include depletion of candidate proteins using siRNA followed by immunocytochemistry to detect nucleolar translocation of RelA and Ni-agarose precipitation of 6HisUb followed by anti-RelA western blot analysis to detect ubiquitination of the protein. We will also use a proteomics approach to identify novel, aspirin-induced, COMMD1 binding proteins. GST-COMMD1 will be isolated from cells +/- aspirin. COMMD1 binding proteins will be identified by SDS PAGE and mass spectrometry of excised bands. Immunocytochemistry, western blot analysis and qRTPCR will be used to analyse the effects of aspirin on components of the complex and the mechanisms by which aspirin causes increased cellular levels of COMMD1.
Objective 2. Identification of the lysine residues of RelA that are ubiquitinated in response to aspirin and the nature of this ubiquitination. Deletion and site directed mutagenesis will be used to identify domains and lysine residues of RelA important for ubiquitination. We will use ubiquitin-mediated fluorescence complementation (UbFC) assays, expression of mutated ubiquitin and linkage specific antibodies, to identify the ubiquitin linkages important for nucleolar translocation of RelA.
Objective 3. Identification of proteins that transport ubiquitinated RelA to the nucleolus. We propose a novel proteomic approach to identify proteins that specifically bind to aspirin-induced, ubiquitinated RelA which involves a combination of isolation of ubiquitinated RelA and stable isotope labelling of amino acids in culture (SILAC). Once binding proteins are identified, techniques outlined in objective 1 will be used to determine their role in aspirin-mediated nucleolar translocation of RelA.

The beneficiaries of this research will be:
1. Academia: As outlined in the academic beneficiaries section, this research will be of considerable and immediate interest to national and international researchers in a number of academic fields including colon cancer prevention nucleolar transport pathways, regulation of cell growth and death by NF-kB and nuclear regulation of RelA. These scientists will benefit from theoretical advancements in the fields, as well as gaining access to the biological tools that will be generated as part of the research and any methodologies developed.
2. Pharmaceutical companies: Uncontrolled NF-kB activity is a contributory factor in a number of common diseases. Therefore, pharmaceutical companies are extremely interested in developments that may lead to novel methods to inhibit this activity. Identification of pathways that cause RelA to translocate to the nucleolus has the very real potential of revealing targets for drugs that manipulate these pathways to switch off NF-kB. As the nucleolar presence of RelA causes apoptosis, the research will also be of interest to companies interested in developing novel anti-cancer agents.
3. Patients and health professionals: Identifying the mechanisms by which aspirin upregulates COMMD1 may have an immediate impact on colorectal cancer patients as it may reveal biomarkers of response in clinical trials of chemopreventative agents. In the long term, understanding the mechanisms by which NSAIDs prevent colorectal cancer may lead to the development of novel agents that could be used to prevent this disease in high risk patients.
4. General public: Media publicity of grant awards and published work will raise awareness of this preventable disease and may prompt the general public to question their risk.