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Protein tyrosine phosphatases as regulators of N-cadherin-mediated tumor cell migration

Final Report Summary - PRENCTUM (Protein tyrosine phosphatases as regulators of N-cadherin-mediated tumor cell migration)

Malignant transformation is often characterized by major changes in the organization of the cytoskeleton, decreased cell-cell adhesion and aberrant adhesion-mediated signalling. The epithelial-to-mesenchymal transition (EMT) is a process during which epithelial cells acquire properties that are characteristic of mesenchymal cells, including disruption of normal cell-cell adhesion and increased motility. These changes play an important role in the transition of epithelial tumours from a benign to an invasive state. Cadherins mediate homophilic cell-cell interactions, and the cytoplasmatic tails of cadherins associate with catenins that link the complex to the cytoskeleton which is necessary for stable adhesions to form. In normal epithelium, E-cadherin forms adherens junctions that participate in contact inhibition of growth. During EMT, expression of the non-epithelial N-cadherin is upregulated, whereas E-cadherin expression is downregulated- a phenomenon known as the cadherin switch. Cadherin switching has been demonstrated in primary tumours as well as in several carcinoma-derived cell lines. Increased expression of N-cadherin has also been detected during melanoma progression and although clinical data has yet to be obtained, N-cadherin has been suggested as a potential target in cancer therapy.

Tyrosine phosphorylation of proteins is an essential component of signal transduction pathways that regulates cell growth, and survival, as well as adhesion, migration and differentiation. The combined functions of protein tyrosine kinases and protein tyrosine phosphatases (PTPs) serve to control cellular phosphotyrosine levels. The role for PTPs as potential tumour suppressors is well established, but more recent findings have demonstrated that several PTPs also are pro-tumorigenic. These findings have prompted the development of drugs acting as selective PTP inhibitors.

Regardless of E-cadherin status, forced expression of N-cadherin is associated with a gain in motility, invasiveness and metastatic ability. A number of studies have shown that signals induced by the family of cell adhesion molecules are partly mediated through activation of FGF receptors. In line with this, expression of N-cadherin in the non-invasive MCF-7 human breast carcinoma cell line increased the migration towards FGF. Comparatively little is known about the regulation of FGFR signalling by PTPs. The stability of E-cadherin-mediated adherens junctions is regulated by tyrosine phosphorylation, and multiple PTPs associate with the E-cadherin complex to stabilize cell-cell adhesion. Whereas the regulation of E-cadherin-dependent adhesion has been thoroughly studied, it is less clear how N-cadherin regulates cell motility. Even less is known about the mechanisms whereby N-cadherin regulates tumour cell motility, but the finding that N-cadherin phosphorylation by the kinase Src is required for transendothelial migration of melanoma cells indicates that interplay between tyrosine kinases and PTPs are important also in tumour cell migration. The notion that not only kinases but also PTPs are necessary for N-cadherin adhesion and function suggests that altered PTP expression or activation will regulate N-cadherin-mediated cell motility and metastasis. To address this possibility, we employed a wide set of in vitro and in vivo cell biological, biochemical and imaging tools to analyse the role of several PTPs in N-cadherin-mediated and FGF directed tumor cell migration and invasion.

The project was split into five main tasks: (1) determining the effects of down regulation of PTPs on cancer cell migration and invasion (2) characterization of FGF receptor phosphorylation and signal transduction after down regulation of PTPs (3) characterization of N-cadherin adhesion complexes (4) characterization of changes in cell morphology and phenotype (5) monitoring tumour growth, invasiveness and metastasis in vivo. Completion of tasks 1 to 4 was necessary in order to commence with task 5 to ensure an ethically sound approach.

These studies revealed a number of important results which add to our knowledge on the involvement of PTPs in cancer cell migration - and invasion. Breast cancer cell lines were modified in order to express N-cadherin or to suppress N-cadherin and were further modified to specifically suppress the expression of several PTPs. By employing cell migration and cell invasion assays we show that cells expressing N-cadherin were more motile and invaded invasion substrate (matrigel) more than cells with lower or no expression of N-cadherin. Importantly, suppression of PTP expression by knock down (kd) techniques revealed that especially the PTPmu, LAR and DEP-1 are important mediators in cells migration and invasion. We find that kd of PTPmu specifically reduced the pro-invasion effect of N-cadherin after cells were stimulated with FGF, the ligand for FGF receptor 1 (FGFR1). Most importantly, when further analysing why PTPmu has this effect we find that suppression of PTPmu affected the phosphorylation status of N-cadherin with a consequent loss of FGFR1 from adhesion complexes and reduction of the activation of the FGFR1 signalling pathway. This points to that the physical interaction between N-cadherin and FGFR1 in the cell membrane, which normally results in prolonged FGFR1 signalling and motile and invasive phenotype, is maintained by PTPmu through dephosphorylation of N-cadherin tyrosine residue(s). In addition, it suggests that this physical link is crucial for FGFR1 stability in the membrane and prolonged activation of the FGFR1 signalling pathway. The in vivo studies from task 5 are still ongoing in order to evaluate the in vitro findings from task 1-4 and confirm the biological importance of PTPmu in a metastatic cancer model. Here we focus predominantly on the metastatic potential of cells lacking PTPmu as compared to cells expressing PTPmu. A pilot study has already shown the possibilities and limitation of techniques required for successful completion of this task and has supplied information for an evaluation of the ethical considerations in connection to the study. Together the results obtained during this Marie Curie Fellowship show that PTPmu is an important player in N-cadherin-mediated cancer cell invasion through its modification of N-cadherin and consequent suppression of FGF receptor signalling. Therefore this tyrosine phosphatase could potentially be a valid therapeutic target for the treatment of cancer metastatic disease.

In terms of the original 5 tasks, tasks 1 to 4 are complete while task 5 is only partially complete; owing to a delay in acquiring definite and conclusive results from tasks 1-4. From an ethical perspective more definitive and conclusive results were warranted prior to initiation of task 5. Although task 5 could not be completed within the project lifespan, important progress was made and it is expected to meet the project goals over the longer term. Scientifically the project has been a success, highlighting the importance of PTPs to the wider biochemical community and providing a basis for further clinical development of potential PTP inhibitors in metastatic disease.