Cell Adhesion-linked Kinases

The Src and FAK non-receptor tyrosine kinases are altered in cancer. They contribute to the migratory phenotype associated with late stage disease, and to changes in tumour cell survival. Our work aims to identify key effectors that link tyrosine kinase-induced migratory and survival signals in cancer cells. One major focus is to understand the spatial and temporal control of complexes between kinases and effectors using fluorescence imaging, and to identify useful bio-markers, as well as novel targets for intervention. This is timely because Src inhibitors have now entered clinical trials here in Glasgow and elsewhere.

Control of Src activation and membrane targeting by RhoB
We have examined the spatial control of Src activation and the nature of intracellular structures that mediate its stimulus-induced transit to the membrane. Src is activated during transit, specifically in RhoB-containing cytoplasmic endosomes associated with the perinuclear recycling compartment (see Figure). Knocking out RhoB, or expressing a dominant-interfering Rab11 mutant protein, suppresses both catalytic activation of Src and translocation of the active kinase to peripheral membrane structures. Src- and RhoB-containing endosomes also harbour proteins involved in actin polymerisation and filament assembly, including Scar1 and mDia2, and newly polymerised actin can associate with these endosomes in a Src-dependent manner. Furthermore, Src controls the actin dependence of RhoB endosome movement towards the plasma membrane. This implies that Src regulates an endosome-associated actin nucleation activity, and we are (in the process of) elucidating the mechanisms involved. This work identifies RhoB as a component of "outside-in" signalling pathways that coordinate Src activation with translocation to membrane receptors, and may be indicative of a more generalrole for RhoB in trafficking membrane-associated proto-oncoproteins to their specific sub-cellular sits of activity (Sandilands et al., Dev Cell, 2004; 7(6):855). In this regard, Rab11-dependent perinuclear recycling endosomes have also been associated with integrin trafficking during cell migration, and it seems likely that the transport of integrins and of their non-receptor tyrosine kinase effectors, are co-ordinately regulated. This has implications for optimal cell spreading on extracellular matrix, and for cell migration. Intriguingly, Rab25 (a close relative of Rab11 that is expressed in epithelial cells), is up-regulated in aggressive ovarian and breast cancers, suggesting that control of intracellular trafficking events may contribute to the malignant phenotype.

Src's special role in epithelial cancer cells
Because Src expression is elevated during acquisition of invasive and metastatic capacity in human cancer cells, we need to understand how Src functions in epithelial cells. We have used a human colorectal cancer metastasis model to show that Src does not necessarily contribute to enhanced growth of metastatic cells. Instead, elevated Src often induces the formation of αvβ1 integrin adhesion complexes, and concomitant de-regulation of E-cadherin membrane targeting, leading to loss of epithelial cell-cell adhesions and a more migratory phenotype (a mesenchymal transition). Interestingly, Src-induced de-regulation of E-cadherin requires integrin signalling, demonstrating interdependence between these two adhesion types (Avizienyte et al., Nat Cell Biol, 2002; 4: 632). Moreover, Src-induced activation of the MEK/ERK/MLCK cascade is a critical determinant of the mesenchymal phenotype, showing that spatially regulated contractile force regulates cancer cell plasticity, mediating a switch between the epithelial- and mesenchymal-like states (Avizienyte et al., Mol Biol Cell, 2004; 15: 2794). In addition, Src co-operates with other malignancy-associated changes in cancer cells to induce the formation of podosomes, actin rich structures that are the sites of invasive protrusion. We are continuing to define spatially-regulated Src activities that control cancer cell invasion.

FAK as a Src effector kinase during oncogenic transformation
Many cell responses that signal through focal adhesion kinase (FAK) are often initiated by integrin-induced auto-phosphorylation of FAK or via phosphorylation by Src family kinases (SFKs). We utilised a multi-site mutant FAK protein that cannot be phosphorylated by upstream SFKs to demonstrate a key role for Src-mediated tyrosine phosphorylation of FAK during: a) the dynamic regulation of actin filaments and cell migration, and b) the survival signalling that permits anchorage-independent growth of transformed cells (Westhoff et al., Mol Cell Biol, 2004; 24: 8113). In cancer cells, FAK is phosphorylated not only by SFKs, but also by other tyrosine kinases recruited to integrin adhesion sites by the Src homology protein interaction domains - SH3 and SH2. The adaptor functions of over-expressed Src can thus contribute to the cancer cell phenotype. However, FAK-tyrosine-925 phosphorylation is Src kinase-dependent and this regulates turnover of integrin adhesions at the rear of migrating cancer cells (Brunton et al., Cancer Research, 2004; in press). We are also examining the regulation and role of tyrosine phosphorylation of p120^ctn, a Src substrate that operates downstream of E-cadherin.

Serum deprived SYF -/- cell re-expressing Src-WT-GFP and myc-RhoB.

FAK deletion impairs tumour progression
FAK expression is elevated, often at the level of gene dosage/amplification, in epithelial cancer cells. Despite this, and the overwhelming evidence that Src is also elevated, a causal link between expression of these kinases and tumour formation or malignant progression is lacking. We have generated a skin-specific conditional knockout mouse in which FAK coding sequences are excised using the Cre-lox system. Skin carcinogenesis experiments revealed that FAK deficiency leads to reduced incidence of papillomas after 7,12-dimethylbenz[a]anthracene (DMBA) treatment. When FAK is knocked out after papillomas have formed, there is inhibition of TPA-induced conversion of papillomas into malignant carcinomas. Mechanistically, loss of FAK is associated with increased apoptosis in both keratinocytes in vitro and in the skin in vivo, particularly in the hair follicle bulge region where the stem cell targets for tumorigenesis reside. FAK's role in survival signalling is thus a determinant of both tumour initiation and progression in mouse skin (McLean et al., Genes and Development, 2004; in press). Together with in vitro studies, our work implies that the Src/FAK tyrosine kinase cascade may be a valuable anti-cancer target. This proof is important because Src inhibitors are now being tested clinically here in Glasgow and elsewhere. We are working with pharmaceutical industry partners to evaluate exciting new Src inhibitors.