Role of the protein tyrosine phosphatase DEP-1 in Src activation and the mediation of biological cell functions of endothelial and breast cancer cells

The implication of protein tyrosine phosphatases (PTPs) in the regulation of cell signalling events and the mediation of cellular functions in response to growth factors such as VEGF has been well-established in the last years. Nonetheless, molecular mechanisms by which PTPs regulate fundamental processes such as angiogenesis are not well-characterized. Expression of the PTP DEP-1 (Density-enhanced phosphatase 1) was reported to increase with cell density and was associated with VEGFR2 dephosphorylation contributing to cell contact inhibition in confluent endothelial cells. We previously demonstrated that DEP-1 attenuates VEGFR2 activity by dephosphorylation of its Y1054/1059 leading to decreased activation of major signalling pathways downstream of VEGFR2 with exception of the Src-Gab1-AKT pathway. Increasing Src activity due to DEP-1-mediated dephosphorylation of its Y529 promotes endothelial cell survival.  The objective of this thesis was to gain a better understanding of the role of DEP-1 in the regulation of the Src activity and of biological responses in endothelial cells.  We identified tyrosine Y1311 and Y1320 in the C-terminal tail of DEP-1 as major phosphorylation sites in response to VEGF. These residues are required for Src activation and mediate the Src-dependent remodelling of endothelial cell junctions inducing permeability, invasion and capillary formation upon VEGF stimulation. We showed that VEGF-induced DEP-1 tyrosine phosphorylation directs DEP-1 specificity towards its substrate Src. Our results thus highlighted for the first time the promoting role of DEP-1 on the angiogenic program in endothelial cells.  In addition to tyrosine phosphorylation, DEP-1 is constitutively phosphorylated on a threonine residue (T1318) proximal to Y1320 in its C-terminal tail suggesting it might be involved in the regulation of Y1320. Indeed, we found that DEP-1 T1318 is phosphorylated, potentially by CK2, and regulates the tyrosine phosphorylation of DEP-1 and its ability to bind to and activate Src. Consistent with this, remodelling of endothelial cell junctions and permeability are impaired in endothelial cells expressing the DEP-1 T1318 mutant. Thus, DEP-1 phosphorylation on T1318 displays a regulatory control over DEP-1 tyrosine phosphorylation and subsequently Src activation and endothelial cell functions in response to VEGF.  Our results demonstrating that DEP-1 promotes angiogenic cell responses in endothelial cells, prompted us to consider a possible involvement of DEP-1 in cancer cells, where Src activation has been linked to cancer progression. Thus, although, DEP-1 is believed to act as a tumour suppressor in different cancer types, we hypothesized that it might also promote Src-dependent functions such as migration and invasion in cancer cells. Interestingly, we found that DEP-1 is higher expressed in more invasive basal-like breast cancer cells than in luminal-like cell lines. Moreover, DEP-1 is implicated in the regulation of Src activity, Src-mediated cell motility and appropriate localization of proteins mediating cytoskeletal organization in basal-like breast cancer cell lines. To further support these results, analysis of a breast cancer tissue microarray revealed that DEP-1 expression is associated with a tendency towards reduced overall survival. Thus, our results provide first evidence for a tumour-promoting role of DEP-1 in breast cancer.  Altogether, the work performed in the context of this thesis revealed that DEP-1 can similarly behave as a promoter of the angiogenic response and of the pro-invasive phenotype in basal-like breast cancer cell lines, most likely due to its ability to activate Src. This suggests for the first time that DEP-1 expression could contribute to tumour progression and the formation of metastases, and as such, represent a potential new target for anti-angiogenic and anti-cancer therapy.

Keyphrases: DEP-1, VEGFR2, Src, Invasion, Angiogenesis, Endothelial cells, DEP-1, VEGFR2, Src, VE-cadherin, Permeability, Invasion, Breast Cancer