The Role and Regulation of Tiam1-Rac Signalling in Bipolar Spindle Assembly

During cell division our two identical sets of chromosomes need to be divided accurately into two daughter cells. This process relies on the mitotic bipolar spindle, a structure composed of microtubules (dynamic cytoskeletal structures) which capture the chromosomes. Formation of the spindle involves two centrosomes, the organising centres for microtubule nucleation. The way the centrosomes move apart to form the spindle when cells enter mitosis is very highly regulated. Scientists have previously shown that one protein in particular, Eg5, is very important for this separation. Drugs which target this protein completely stop the centrosomes separating and cause cells to arrest in mitosis with 'monopolar' spindles.

We have recently found that a protein (Tiam1) and its substrate (Rac), which were previously known to be important in regulating the migration of cancer cells, are also important for regulating this centrosome separation process in mitosis. Tiam1 and Rac produce a force which counteracts the force of Eg5 during spindle formation. We have shown that this balance of forces is necessary to allow the chromosomes to become efficiently captured by the microtubules. We have also found that Tiam1 interacts with the MPF, an important mitotic complex (which also regulates the function of Eg5), and that regulation of Tiam1 by MPF is important for the function of Tiam1 in regulating centrosome separation. However, we currently do not know the processes downstream of Tiam1 and Rac in this process, and it is likely that a number of other, unknown proteins are involved.

Understanding the molecular mechanisms involved in the process of cell division is extremely important for cancer research. Firstly, it is known that de-regulation of accurate division can cause cancer by the loss or gain of important genes. Secondly, many current treatments target the process of cell division because cancer cells divide more rapidly than normal cells, so knowing which proteins regulate this process allow us to predict how cancers will respond to certain chemotherapies.

The force produced by Tiam1 and Rac means that when one of these proteins is absent, or inhibited, cells can more readily 'escape' inhibition of Eg5. Currently, Eg5 inhibitors are in clinical trials for cancer treatment, so looking more closely at the mechanisms of this signalling pathway is important for being able to predict how cancers will respond to this new class of cancer therapeutics.

The way that we think Tiam1 and Rac exert their effect on centrosome separation, namely by regulating microtubules, also means they can alter cells' responses to another class of cancer therapeutics, that target the microtubules to kill cancer cells. We plan to investigate this further, by repeating our experiments in cancer cells to see whether Tiam1 and Rac could be a biomarker for treatment with these drugs.

Because of the potential importance of this signalling pathway for tumourigenesis and cancer therapy, we aim to further characterise the signalling pathway, and our previous work means that we already have a number of tools available to achieve this. Part of this project involves looking more closely at the regulation of microtubules by Tiam1 and Rac. Another important aspect of this work is to identify the proteins which work together with Tiam1 and Rac to regulate centrosome separation. We can take two different approaches to this; firstly, we can use the tools we have to initiate a screen for new potential interacting proteins, and secondly we can use a candidate approach to individually test candidates from our previous experiments, or the literature, which seem likely to be involved.

This work will not only further our understanding of the important process of centrosome regulation in mitosis, but, as described above, may have important implications for tumourigenesis and current cancer therapies.

Previously we showed that Tiam1 and Rac regulate centrosome separation and mitotic spindle function. Now, our latest data imply that phosphorylation of Tiam1 on S1466 by CDK1/ cyclin B is required for these functions. We have generated S1466A and S1466D mutants and shown these to be functionally inactive and effective respectively, and have produced a phospho-specific antibody to this site which shows a CDK1-dependent increased signal in mitosis. Our experimental plan takes advantage of the knowledge and tools we have generated so far, beginning with the use of our phospho-specific antibody to analyse the spatial and temporal dynamics of S1466 phosphorylation by immunofluorescence and/or biochemistry. We also plan a series of experiments to examine further the role of Tiam1 and Rac in regulating microtubule stability, and will use our phospho-mutants to directly link the phosphorylation of Tiam1 to this mechanism. An important part of our experimental plan is to identify other proteins involved in the mitotic Tiam1/ Rac signalling pathway; specifically, proteins which preferentially bind to the S1466 phosphorylated form of Tiam1. For this we will adopt both a global approach, using our S1466D and S1466A mutants in a mitosis-specific mass spectrometry experiment, and a candidate approach, by selecting particular proteins from the literature and/ or previous interactor screens as likely candidates. Candidate proteins will be tested firstly for potential interactions with Tiam1 in transient co-immunoprecipitation experiments, then functional experiments will be performed on successful candidates using a combination of techniques. Finally, we aim to take advantage of our close relationship with the breast pathology lab of Prof. Goran Landberg to investigate the phosphorylation status of Tiam1 in breast tumours, as well as investigate the effect of modulation of this signalling pathway on the sensitivity of breast cancer cells to treatment with anti-microtubule drugs.

The proposed research explores the fundamental mechanisms of cell division and how these are deregulated in tumourigenesis. Immediate beneficiaries are other academics researching in this field as the further knowledge generated will allow a more accurate, more detailed model to be generated that can lead to their own data being assimilated into this model as well as new hypotheses being conceived. They will also benefit from novel reagents and analytical techniques generated during the course of the research that will be distributed to the research community. A potential next group of beneficiaries include the pharmaceutical industry who seeks new targets for anti-cancer therapeutics, as well as biomarkers for diagnosis and prognosis, personalizing medicine and assessing treatment efficacy. In particular, the proposed research involves assessment of the effect of modulation of this signalling pathway on the sensitivity of cancer cells to anti-microtubule drugs (AMDs) which are currently used for cancer treatments as well as Eg5 inhibitors that are currently under clinical evaluation. This knowledge may identify our proteins of interest as potential biomarkers for treatments with these groups of cancer therapeutics. Another group of potential beneficiaries therefore include patients whose treatment could be improved due to the knowledge generated during our research, potentially resulting in improved survival or decreased pain and suffering. Additionally, this research will increase the skill and knowledge base of the lab and would enhance the ability of the lab to recruit and train scientists for academia, public sector and industry, thereby benefiting the economy. Moreover, the continuing scientific training and career development of the co-investigator benefits herself as well as her future employer and the economy.