The Wilms' tumour suppressor WT1 is involved in endothelial cell proliferation and migration: expression in tumour vessels in vivo

Abstract

Vascularization is an important step in tumour growth. Although a variety of molecules, for example, VEGF, ETS-1 or nestin have been implicated in tumour angiogenesis, the molecular mechanisms of vessel formation are not fully characterized. We showed that the Wilms' tumour suppressor WT1 activates nestin during development. Here we tested whether WT1 might also be involved in tumour angiogenesis. Endothelial WT1 expression was detected in 95% of 113 tumours of different origin. To analyse the function of WT1 in endothelial cells, we used an RNAi approach in vitro and showed that inhibition of WT1 reduces cell proliferation, migration and endothelial tube formation. On a molecular level, WT1 silencing diminished expression of the ETS-1 transcription factor. WT1 and ETS-1 shared an overlapping expression in tumour endothelia. The ETS-1 promoter was stimulated approximately 10-fold by transient co-transfection of a WT1 expression construct and WT1 bound to the ETS-1 promoter in chromatin immunoprecipitation and electrophoretic mobility shift assays. Deletion of the identified WT1-binding site abolished stimulation of the ETS-1 promoter by WT1. These findings suggest that transcriptional activation of ETS-1 by the Wilms' tumour suppressor WT1 is a crucial step in tumour vascularization via regulation of endothelial cell proliferation and migration.

WT1 gene silencing by aerosol delivery of PEI–RNAi complexes inhibits B16-F10 lung metastases growth

“…The reduced number of blood vessels in the treated samples suggests that silencing of WT1 inhibits angiogenesis. This is consistent with the observation that WT1 activates the ETS-1 gene, which is important for tumor vascularization through regulation of endothelial cell proliferation and migration, 50 and with studies about WT1 transcriptionally regulating vascular endothelial growth factor expression. These molecular mechanisms may be responsible for the role of WT1 in angiogenesis and cancer progression.…”

The Wilms’ tumour suppressor Wt1 is a major regulator of tumour angiogenesis and progression

“…Endothelial cell c-kit expression has been described, both, in in vitro 36 and in vivo studies of human 37 and mouse 38 tissues. Western blots confirmed downregulation of Ets-1, which we already identified as a WT1 target in human endothelial cells 9 , and of c-kit and Pecam-1 upon knockdown of Wt1 (Supplementary Figs 13a and 14). Wt1 knockdown decreased migration and proliferation of mouse endothelial cells, in agreement with our study on their human counterpart 9 .…”

“…Western blots confirmed downregulation of Ets-1, which we already identified as a WT1 target in human endothelial cells 9 , and of c-kit and Pecam-1 upon knockdown of Wt1 (Supplementary Figs 13a and 14). Wt1 knockdown decreased migration and proliferation of mouse endothelial cells, in agreement with our study on their human counterpart 9 . Overexpression of c-kit did not significantly change proliferation, but increased migration of C166 cells compared with controls.…”

“…We investigated vascular and stromal WT1 expression in a series of 80 human cancers from seven tumour types (lung, breast, ovary, colon, pancreas, prostate carcinoma and melanoma). As described, WT1 was strongly expressed in a mostly nuclear, sometimes cytoplasmic pattern in tumour vessels 9 , but also in stromal cells (Fig. 1).…”

“…We found earlier that the Wilms' tumour suppressor WT1 is highly expressed in vessels of most human tumours, but not in healthy adjacent tissues 9 . WT1 encodes a zinc-finger transcription factor with crucial functions in development and has originally been identified as a tumour suppressor 10 .…”