Cross-talk between Ajuba and Rac signalling in the stabilization of cadherin adhesion

Cell-cell adhesion is a key event in the life of different cell types and is essential for their organization into higher ordered structures such as blood vessels, lung, skin, muscle, etc. Conversely, de-regulation of cell-cell contacts is found following bacterial infection, would healing or initiation of tumour invasion. We are interested in understanding how cells control their ability to glue to their neighbours as opposed to scatter into surrounding tissues. We identified a molecule, Ajuba, which plays a key role to strengthen cell-cell adhesion: in the absence of Ajuba, cell-cell aggregation is greatly reduced. Our unexpected finding is that Ajuba can manipulate the function of the small GTPase Rac, which is required to stabilize cell-cell contacts. In this proposal, we will address the mechanisms via which crosstalk between Ajuba and Rac leads to stronger cell-cell adhesion. Understanding the mechanisms via which Ajuba/Rac stabilize junctions will highlight potential strategies to interfere with tumour cell invasion and metastasis. Suitable strategies to stall metastatic growth will have important implications for the quality of life of cancer patients.

We have evidence that Ajuba plays a role in Rac dynamics, and conversely, Rac/PAK1 signalling modulates Ajuba-dependent junction stabilization. We will address in three Work Packages:

1 - how Ajuba modulates Rac dynamic. We postulate that Ajuba interaction may prevent Rac.GTP hydrolysis or facilitate recruitment of an exchange factor to junctions to activate Rac. Rac GTP loading and intrinsic hydrolysis will be addressed in the presence/absence of wild-type Ajuba or its phosphomimetic (T172D) or non-phosphorylatable (T172A) mutants. Ajuba mutants unable to interact with Rac will be made and tested in vivo for junction stabilization and rescue of Rac activation. As depletion of DOCK180, a Rac GEF, phenocopies Ajuba RNAi, a potential role for DOCK180 in Ajuba-dependent Rac activation by cadherins will be addressed.

2 - how Ajuba reorganizes actin at cadherin complexes. We will address the relative contribution of Ajuba-dependent actin polymerization versus bundling for cadherin stabilization and how Rac/PAK1 signalling influences these two activities. Mutational analysis will identify the F-actin binding sites on Ajuba responsible for polymerization or bundling and the efficiency of these mutants to strengthen cadherin adhesion. The existence of ternary complexes Rac/Ajuba/Actin or Rac/Ajuba/alpha-catenin will be studied as a mechanism to integrate cadherin complexes with actin cytoskeleton.

3 - the relevance of Ajuba in Rac/PAK1 signalling during tumour progression will be tested. Up-regulation of Ajuba protein and phosphorylation levels will be investigated in tumour tissue microarrays and cell lines (primary and metastatic site) and correlated with malignancy and clinical outcome. We will test Ajuba sub-cellular localization in tumours/cell lines (nucleus, junction or focal adhesion) and which cellular process it regulates downstream of Ras/Rac hyper-activation during transformation (proliferation, invasion and/or junction disassembly).

Our proposal has impact for public health, improving quality of life and UK economic competitiveness. Because epithelial cells are present in many body sites, understanding how their function is maintained has profound implications for the physiology of many different organs. Epithelial cells require constant signalling from junctions to maintain their polarized, differentiated phenotype. Improving knowledge on how cadherin-dependent junctions are maintained will provide potential mechanisms for therapeutic intervention in different clinical settings.

The biomedical aspect of the research may lead to applications for commercial exploitation, and hence increase economic competitiveness in the UK. Because of the dual aspect of the proposed work, including fundamental research as well as its biomedical applications, the project will support knowledge and technology translation and increase the economic competitiveness. The core principles investigated here (interplay between adhesion receptors and signalling) are frequently perturbed in different pathologies.

Cancer:
Over 80% of human tumours originate from epithelial cells and thus, specific knowledge on epithelial processes is relevant to identify processes amenable to intervention. Most deaths of cancer patients may be prevented by reducing dissemination/growth of metastasis or damage to distant organs by malignant lesions. Reduced cell-cell adhesion in tumours impairs epithelial differentiation and appropriate localization of signalling molecules and tumour suppressors. Understanding the mechanisms via which cell-cell adhesion is de-regulated during tumour progression will provide therapeutic strategies to ameliorate cell de-differentiation and detachment from solid tumours and metastasis. Such strategies have considerable clinical implications as adjuvant therapy, to sustain stable tumour mass of non-responsive tumours or tumours inaccessible to surgery.

Other diseases:
(a) A "leaky" epithelium underlies the causes of many chronic diseases such as asthma, atopic dermatitis, food intolerance/allergy, and chronic infections. When the tightness of epithelial sheets is compromised are defective (including cadherins and tight junctions), exogenous components such as airborn antigens (skin, lungs) or breakdown food products (intestines) are able to leak through the epithelium and stimulate the underlying immune system.
(b) Bacterial infections: different pathogens highjack adhesion receptor signalling to mediate their internalization. An example specifically involving E-cadherin is invasion of the pathogen Listeria monocytogenes, a serious infection for infants and immuno-compromised individuals. Another example is the intricate interplay between Helicobacter pylori and E-cadherin in stomach and gastric epithelia, a risk-factor in development of ulcers and gastric cancers.
To increase the impact of future therapies to specific cohorts of cancer patients, our work will define the type of tumour in which interfering with the cross-talk Rac>PAK1>Ajuba is most beneficial. Rac signalling is often up-regulated in different and, on their own right, Rac and PAK1 play an essential role in numerous diseases. Ajuba mRNA is increased in different tumours some of which are previously reported subsets that show Rac and PAK up-regulation. In addition, Ajuba gene is located in 14q11.2, a breakpoint site for leukemia mantel cell leukemia. Thus, identifying novel regulatory circuitry that controls Rac/PAK1 signalling will provide insights towards the design of small molecules to be used for translational purposes.