Regulation of the Rac activator Tiam1 by ubiquitylation

RAS proteins are master regulators of biochemical pathways involved in the transformation of cells from normal to cancer. Their function is abnormal in most human tumours. Investigation of the pathways activated by RAS proteins has identified molecules which can be targeted by drugs to inhibit tumour growth and spread.
RAC is a protein structurally similar to RAS which indeed mediates the cancer-inducing properties of RAS. RAC activates a wide range of processes such as cell migration and invasion. RAC cycles between two different states 'on' or 'off' controlled by two families of proteins known as Guanine nucleotide Exchange Factors (GEF) and the GTPase activating proteins (GAPs) respectively. In its active or switched 'on' state RAC is able to bind various other proteins to elicit consequent responses in the cell.
The T-cell lymphoma invasion and metastasis 1 (TIAM1) protein is a GEF specific for the activation of RAC and is itself required for tumour formation. However, TIAM1 through RAC can also stimulate the adhesion of cells and potentially slow down the spread of cancer. Unpublished data from our lab now indicates that TIAM1 levels can be reduced via its interaction with another protein- HUWE1- which labels TIAM1 for degradation. We hypothesize that HUWE1 may accumulate in cancer cells in order to reduce TIAM1 to an amount that still allows tumours to develop but can not inhibit cancer spreading.
There are three main objectives to this proposal. Firstly we will define the parts of these two proteins that allow physical interaction. Secondly, using this information we will then make 'mutant' TIAM1 proteins which cannot be degraded and by replacing normal TIAM1 with mutant TIAM1, we will functionally assess the importance of TIAM1 degradation during processes involved in cancer growth and spreading. Thirdly, we plan to use two different mouse models where the expression of TIAM1 and HUWE1 can be genetically altered, to investigate the effect of TIAM1 and HUWE1 modulation on tumourigenesis. Finally, to confirm the importance of the above findings to the field of cancer research we plan to analyse the expression of TIAM1 and HUWE1 in human tumour samples. We envisage that the knowledge we learn from these studies will increase our understanding of how abnormal RAS function results in cancer and improve the design of drugs to treat cancer.

RAS genes are frequently mutated in human cancer. The proteins they encode drive cell proliferation, survival, invasion and metastasis. To develop therapeutic agents targeting RAS function, focus has been applied to its downstream effector pathways. RAC1 is a key mediator of RAS transformation, as demonstrated by the resistance of Rac1 knockout mice to develop various Ras-induced tumours. TIAM1 is a selective activator of RAC that couples RAC and RAS activation. Like Rac1, Tiam1 is required for Ras-induced tumour formation in mice. However, mouse tumours lacking Tiam1 progress more frequently to malignancy, potentially due to TIAM1-RAC's ability to stimulate intercellular adhesion and thereby suppress invasion. Nonetheless, it is apparent that TIAM1 can also promote single cell motility and invasion. To target therapeutically the TIAM1-RAC signalling module requires increased understanding of its diverse cellular roles and also of its intricate spatio-temporal control. In unpublished data, we have identified an interaction between TIAM1 and the E3 Ubiqutin ligase HUWE1 and shown that TIAM1 is ubiquitylated by HUWE1 and degraded by the proteasome during stimulation of cell migration by the cytokine HGF/SF. HUWE1 is overexpressed in a spectrum of human tumours and this has been correlated with malignant progression. We hypothesise that through spatio-temporal regulation of TIAM1 expression, HUWE1 diverts TIAM1-RAC from promoting intercellular adhesion, to allow malignant progression. We now propose to map the interaction sites of TIAM1 with HUWE1 and identify the specific residues that are ubiquitylated. We then propose to determine whether HUWE1 regulates TIAM1 expression in a panel of cancer cell lines and whether manipulating the ubiquitylation of TIAM1 by HUWE1 impacts on cancer cell motility, invasiveness, proliferation or survival. Finally, we shall address the significance of the HUWE1-TIAM1 interaction for tumourigenesis using mouse tumour models.

This proposal focuses on the importance of the ubiquitylation of the RAC guanine nucleotide exchange factor TIAM1 for epithelial cell migration and invasion and potentially proliferation and its perturbation during tumourigenesis. Immediate beneficiaries from this research would be other academics working in this and related fields, as the further knowledge generated will allow a more detailed and accurate model of tumourigenesis to be generated, that could lead to further novel hypotheses being conceived. They will also benefit from the reagents and analytical techniques generated and developed during the course of the research that will be distributed to the research community. A potential next group of beneficiaries include pharmaceutical/biotech companies who seek new targets for anti-metastatic therapeutics, as well as novel biomarkers for diagnosis and prognosis, personalizing medicine and assessing treatment efficacy. The knowledge derived from these studies may identify our proteins of interest (TIAM1, HUWE1 and c-MET) as potential biomarkers for treatments specifically developed to inhibit the metastasis of cancer cells from the primary tumour to secondary sites in cancers that derive from an epithelial origin such as lung, breast and colorectal carcinomas. The terrain of drug discovery is also being transformed by emerging technologies (such as stapled peptides and fragment based drug discovery) which is widening the repertoire of molecular classes that can be effectively drugged. Conceivably HUWE1 or the HUWE1-TIAM1 interface could be targeted in future. A thorough validation now of the significance of this interaction in tumourigenesis would justify this undertaking. 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.