Background

Endothelial cell migration and cell-cell junction formation are key stages in the formation of new blood vessels. We and others have previously demonstrated that these processes are regulated by the Rho family of small GTPases. Activation of Rho GTPases is mediated by guanine exchange factors (GEFs).  The Dock family of GEFs consists of 11 members. However, to date none of these proteins have been previously implicated in tumour angiogenesis. GEFs can be targeted by small molecule inhibitors and therefore they may constitute therapeutic targets in cancer.

Method

In order to identify what GEFs are required for angiogenesis, we have conducted an siRNA-based screen in a three-dimensional tissue culture angiogenesis system. We are studying the roles of “hits” indentified in these screens in the tissue culture angiogenesis model using timelapse microscopy, and in an in vivo model that employs retroviral transduction to achieve knockdown of proteins of interest in the tumour vasculature.

Results

We demonstrate that Dock4 is a Rac1 GEF in endothelial cells downstream of VEGF signalling. Dock4 is essential for cell-cell adhesion and sprouting in the tissue culture angiogenesis model. Knockdown of Dock4 inhibits endothelial cell cluster formation that normally precedes sprouting. Tubules form as result of head to tail cell-cell adhesion and they are thinner. In the absence of Rac1 or Dock4 tubules show reduced sprouting. To gain an understanding into the role of Dock4 in tumours in vivo, we have utilized ecotropic retroviruses to deliver shRNAs to host-derived vasculature in BE xenograft tumours. We show that Dock4 knockdown inhibits the formation of functional lumens in tumours in vivo.

Conclusion

Dock4 is a Rac1 GEF downstream of VEGF signalling required for cell-cell adhesion and sprouting, and lumen formation in vivo. Dock4 knockdown in endothelial cells compromises the generation of functional vessels in tumours.