Recent Progress 2015
Tumour initiation and progression result from the inappropriate activity of intracellular signalling cascades. RHO-like GTPases are molecular switches in signalling pathways that regulate the organisation of the actin and microtubule cytoskeletons, junctional complexes, and extracellular matrix attachments, as well as regulating gene transcription. In this way, RHO proteins influence cell morphology, adhesion, motility, as well as cell cycle progression and survival. Data has emerged to directly implicate RHO proteins in tumourigenesis. We investigate the mechanisms by which certain regulators of the RHO-like GTPase RAC control cell cycle progression and cell‒cell adhesion, how their activities, as well as activity of RAC itself, are controlled and how deregulated RAC signalling contributes to tumourigenesis.
RAC cycles between a GDP- and a GTP-bound state. When GTP-bound, it interacts with various effector molecules that elicit downstream responses, notably including actin cytoskeletal reorganisation. Multiple mechanisms control RAC activity including control of nucleotide binding and hydrolysis by Guanine nucleotide Exchange Factors (GEFs) and GTPase Activating Proteins (GAPs) respectively, regulation of subcellular localisation, modulation of RAC protein levels, and post-translational modification including isoprenylation and as we and others have recently demonstrated, ubiquitylation and SUMOylation (Castillo-Lluva et al., Oncogene. 2013; 32: 1735; Castillo-Lluva et al., Nat Cell Biol. 2010; 12: 1078).
RHO GEFs are typically large proteins harbouring multiple protein‒protein interaction domains. Besides stimulating guanine nucleotide exchange, GEFs act as molecular scaffolds targeting RHO molecules to particular subcellular locations and increasing the local concentration of selective effector molecules, thereby influencing downstream processes. Through influencing selectivity in RHO signalling, GEFs perform non-redundant signalling roles which can confer a significant involvement in tumourigenesis. TIAM1 is a guanine nucleotide exchange factor that selectively activates RAC. Mice deficient for Tiam1 are resistant to the formation of skin tumours induced by chemical carcinogens which target H-Ras and the few resulting tumours grow very slowly (Malliri et al., Nature 2002; 417: 867). Thus TIAM1, we infer, plays a unique role in mediating RAS transformation, which the cell signalling group is currently elucidating.
TIAM1-RAC signalling regulates bipolar spindle assembly, chromosome congression and mitotic progression dependent on phosphorylation of TIAM1 by Cyclin B/CDK1.
To better understand the role of TIAM1 in promoting tumour growth we have examined its role in the cell cycle. Previously, we revealed that TIAM1 and RAC localise to centrosomes during prophase and prometaphase, and TIAM1, acting through RAC, ordinarily retards centrosome separation. TIAM1-depleted cells transit more slowly through mitosis and display increased chromosome congression errors. Significantly, suppression of the microtubule motor Kinesin-5/Eg5 in TIAM1-depleted cells rectifies not only their increased centrosome separation but also their chromosome congression errors and mitotic delay. These findings identified TIAM1-RAC signalling as an antagonist of centrosome separation during mitosis and demonstrated its requirement in balancing Eg5-induced forces during bipolar spindle assembly (Woodcock et al., Curr Biol. 2010; 20:669). Subsequent to this study, we have found that TIAM1 is phosphorylated by Cyclin B/CDK1 at the onset of mitosis. We have now mapped the dominant phosphorylation site on TIAM1 and by creating a non-phosphorylatable mutant demonstrated that while not required for TIAM1 localisation to centrosomes, phosphorylation is essential for its role in regulating centrosome separation. Currently we are investigating the mechanism by which phosphorylation of TIAM1 influences its role at centrosomes and have delineated a novel function of phosphoTIAM1 in stimulating the activity of an important RAC effector that we also implicate in centrosome separation.
Figure 1. In benign epithelial cells, TIAM1 localises to cell‒cell junctions where it stimulates cell adhesion. Cytokines like HGF promote the migration and invasion of epithelial cells by triggering degradation of TIAM1 by HUWE1. Without TIAM1 at cell‒cell junctions, the junctions disassemble giving cells an opportunity to disaggregate. Disassociation of epithelial cells can enhance their motility and invasiveness.
TIAM1 antagonises malignant progression.
Despite their slower growth, tumours arising in Tiam1-deficient mice progress more frequently to malignancy (Malliri et al., Nature 2002; 417: 867). One mechanism by which TIAM1 and RAC suppress malignant progression is through promoting cell–cell adhesion. We recently identified β2-syntrophin, a component of the dystroglycan adhesion complex, as a TIAM1 binding partner. Our study (Mack et al., Nat Cell Biol. 2012; 14: 1169) unearthed a novel role for this complex in promoting tight junction formation and the establishment of apicobasal polarity through the generation of a RAC activity gradient in the membrane region encompassing these junctions.
Malignant progression can entail the loss of cell-cell adhesion. The oncoprotein Src, a non-receptor tyrosine kinase, targets adherens junctions (AJ) for disassembly. Previously, we showed that Src phosphorylates TIAM1 inducing its cleavage by Calpain and its depletion from AJs. Abrogating TIAM1 phosphorylation by Src suppressed AJ disassembly (Woodcock et al., Mol Cell 2009; 33: 639).
In a recently published study, we have now found that TIAM1 is ubiquitylated and degraded upon treatment of cells with hepatocyte growth factor (HGF), a cytokine that is abundant in cancer and believed to promote invasion of cancer cells. We have mapped the ubiquitylation site on TIAM1 and also identified the responsible E3 ligase as being the HECT family member HUWE1. Moreover, we show that interfering with TIAM1 ubiquitylation by depleting HUWE1 or mutating the ubiquitylation site retards the scattering and invasion of cells through delaying AJ disassembly. HGF and HUWE1 are abundant in lung cancer. We show that HUWE1 and TIAM1 expression are inversely correlated in lung cancer specimens. Significantly, we demonstrate that HUWE1 promotes lung cancer invasion by degrading TIAM1 (Vaughan et al., 2014). Potentially, pharmacological agents capable of disrupting the HUWE1-TIAM1 interaction could also antagonise the invasion of lung and other cancer cells, reducing the risk of metastasis.