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The protein interaction and cancer research labLab group photo
Lab head
Lab members
Aileen Heal
Annaloes Mensinga
Chayanica Nasa
Christina Lackmann
Linda Hii
ResearchReceptor Tyrosine Kinase signalling and function in cancerThe principle research focus of the Lackmann lab concerns molecular mechanisms underlying cell motility and cell positioning. We are particularly interested in the notion that the de-regulated emergence of embryonal cell-guidance programs during cancer progression and participate in tumour invasion and metastasis. The largest family of receptor tyrosine kinases, Eph receptors (Ephs), together with their cell-surface ‘ephrin’ ligands co-ordinate the movement and positioning of cells and cell layers in multicellular organisms, in particular during development. Their unscheduled expression and function in the adult contributes to tumour progression, promoting neovascularisation, invasiveness and metastasis.
The current research emphasis of the lab is to understand Eph-mediated cell positioning in tumour cells and to dissect the molecular architecture of the underlying signalling complexes, as basis for design and evaluation of potentially pharmaceutical reagents that can be used to target Eph-positive tumours. We pursue an integrated research strategy, elucidating Eph/Eph contacts by functional mutagenesis and crystallography (together with D. Nikolov, New York), analysing signalling clusters by confocal microscopy and fluorescence life time imaging (together with P. Bastiaens, Heidelberg), and dissecting signalling pathways by forward genetics.
Eph signalling clusters assemble through coordinated tethering of three distinct, high- and low-affinity Eph/ephrin contact surfaces, as well as receptor oligomerisation via membrane-proximal Eph domains. The complex of Eph/ephrin tetramers is sufficient as nucleus for “polymerisation”, whereby the final size of the cluster will depend on the local Eph and ephrin density. (Himanen et al., 2001, Smith et al., 2004, Day et al., 2005). Within minutes of Eph clustering, CrkII and RhoA-facilitated contraction of the cytoskeleton leads to tumour cell rounding, detachment and increased motility (Lawrenson et al., 2002). Concurrently, Eph-mediated endocytosis of ephrin-A5 leads to its lysosomal translocation and degradation (Wimmer-Kleikamp et al., 2004) and signal termination, suggesting a potential route for the delivery of a cytotoxic cargo into Eph+ive tumour cells. On going research projectsBasic researchThe function of Eph signalling in tumour progression. In addition to their role modulating tumour cell adhesion, motility and invasiveness, considerable evidence demonstrates an active role of Ephs and ephrins in neo-vascularisation during normal and pathological conditions. Interestingly, Eph A receptors seem to be active only in adult angiogenesis and are have been over-expressed in lung, kidney and gastric tumour vasculature, and dominant-negative, soluble EphA2 or A3 proteins have been used to control tumour angiogenesis and progression in vivo. In ongoing studies we are using in-vitro and in-vivo cancer models to examine the role of Eph-driven cell positioning in tumour invasion, neovascularisation and metastasis. In parallel, together with A. Scott (Melbourne) and A. Boyd (Brisbane) we are examining Eph protein and RNA expression patterns in the tumour mass and vasculature of malignant melanoma, lung, kidney, brain and colon cancers to identify cancers that can be optimally targeted with Eph-specific anti-cancer agents. Elucidation of the molecular switch used by Eph receptors to trigger either cell-cell adhesion or repulsion. It seems plausible that cell biological outcomes rely on the composition of Eph signalling complexes, and we demonstrated recently that their assembly and lateral expansion involves ephrin-independent recruitment of Eph receptor molecules (Wimmer-Kleikamp et al., 2004). Our finding, of lateral signal progression by receptor self-association as molecular mechanism that triggers Eph functions, suggests that the configuration, dynamics and make-up of Eph signalling clusters on a cell membrane needs to be unravelled to appreciate the regulation of biological outcomes, such as cell-cell adhesion and repulsion. We are addressing this concept in ongoing studies with T. Pawson (Canada) and P. Bastiaens (Germany), investigating the composition and signalling crosstalk of Eph clusters in various cellular settings. The apparent paradox, that cell-cell repulsion is elicited by cell surface-tethered ephrin ligands and Eph receptors. On first sight it seems illogical that high-avidity complexes between Ephs and ephrins on opposing cell membranes result in repulsion of the two interacting cells. We discovered that the intact Eph/ephrin complex is recognized by a transmembrane metalloprotease, ADAM10. A specific binding pocket of ADAM10 orients the protease domain towards the complex for efficient cleavage of receptor-bound ephrin (Janes et al., 2005). This specificity for a ligand/RTK complex is interesting, as ADAM10 is also the protease responsible for EGFR trans-activation, a pathway directly implicated in the pathology of human cancers.
In ongoing studies we examine now the recognition specificity of ADAM10 for other targets and elaborate the role of Eph kinase activity in modulating ADAM10 function. Using integrated structure/function analysis of the ADAM/Eph/ephrin complex (together with D. Nikolov, USA), we establish strategies that will allow the modulation of ADAM-function during tumour progression. Phosphatase regulation of Eph RTK activities. Protein tyrosine phosphatases (PTP) are essential in modulating Eph activity, and in particular LMW-PTP and SHIP-2 have been implicated in regulating Eph functions in tumour cells. Interestingly, PTP’s are regulated by reversible inactivation through reactive oxygen species (ROS), generated as a consequence of growth factor receptor activation or UV radiation. In collaboration with P. Chiarugi (Italy) and T.Tiganis (Melbourne) we are characterising PTPs modulating EphA3 function, in particular in the context of in tumour cell adhesion and motility, aiming to elaborate a potential link between UV radiation, PTP inactivation and modulation of melanoma cell invasiveness. Translational research
In parallel to basic research, we are leading a translational research and development program together with the Tumour Targeting Program at the Ludwig Institute for Cancer Research (A. Scott), the Leukemia Foundation Lab at QIMR (A. Boyd) and a BioPharma partner (KaloBios, Palo Alto, USA), to advance EphA3-specific reagents for clinical use. A soluble form of ephrin-A5 and an anti-EphA3 monoclonal antibody effectively target EphA3-positive, solid tumours or leukemias xenografts. Moreover, binding of the anti-EphA3 antibody to cell surface EphA3 mimics ephrin-A5-triggered activation and in combination with ephrin-A5 dramatically accelerates ephrin endocytosis and lysosomal translocation (Vearing et al., 2005). We are pursuing the humanisation of IIIA4 as clinical tumour targeting reagent, and are exploiting the unique synergy between the antibody and ephrinA5 for the design a targeting construct with potentially accentuated specificity for EphA3-positive tumour cells. The design, evaluation and production of these reagents will provide a platform of tumour targeting reagents with considerable potential for the treatment of metastatic cancers.
Selected publications
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