Biological function of the proteolytic processing of vascular endothelial growth factor-D in cancer

© 2012 Dr. Nicole Cayley HarrisThe Vascular Endothelial Growth Factors are a family of secreted homodimeric glycoproteins and primary regulators of angiogenesis, the growth of new blood vessels, and lymphangiogenesis, the growth of new lymphatic vessels. The mammalian family consists of VEGF, PlGF, VEGF-B, VEGF-C and VEGF-D and they exert biological effects in tissues by binding and activating their cognate receptors, VEGFR-1, VEGFR-2 and VEGFR-3, expressed on endothelial cells. VEGF-C and VEGF-D comprise a subset of the VEGF family as they are initially synthesised as precursor proteins composed of an N- and C-terminal propeptide flanking the central VEGF homology domain, which contains receptor binding sites. Proteolytic processing of these propeptides liberates the mature form consisting of homodimers of the VEGF homology domain, with enhanced affinity for receptors. VEGF-D binds VEGFR-2 and VEGFR-3, and when expressed in tissues promotes blood and lymphatic vessel growth. In animal models of cancer VEGF-D expression induces lymphangiogenesis, increasing lymph node metastasis, and stimulates tumour angiogenesis enhancing tumour growth. VEGF-D can be expressed in a range of prevalent human cancers and this expression correlates with lymph node metastasis and shorter overall patient survival in studies of breast, colorectal and ovarian cancer. Previous analysis of full-length VEGF-D in vivo demonstrated variability in the angiogenesis and lymphangiogenesis induced in tissues, however, processing was not blocked in these model systems so partially and fully processed VEGF-D may have contributed to the effects observed. The activity of full-length VEGF-D, which has not been processed, is not known. In addition, the bioactivity of partially processed VEGF-D and the effect that cleavage of the N- or C-terminal propeptides has on the biology of the molecule has not been defined. Further, it is not known if proteolytic processing is absolutely required for VEGF-D to promote tumour growth and spread. With the aim of determining the effects of the proteolytic processing of VEGF-D on its biological function and action in cancer, a form of VEGF-D was created with mutations at known cleavage sites, effectively blocking processing of both propeptides. Further, to explore the effects of partial processing and the role of the propeptides, additional mutants were generated in which only the N- or only the C-terminal propeptide were deleted and the cleavage site of the remaining propeptide was mutated to block processing. The research described in this thesis demonstrates that each processing mutant displays unique receptor binding and activation profiles, and activity in in vitro assays. In addition, processing of the N- and C-terminal propeptides is a critical determinant of the interactions of VEGF-D with neuropilins and heparin-containing proteoglycans. Tumour xenograft experiments showed that completely blocking VEGF-D processing significantly reduced tumoural angiogenesis and lymphangiogenesis, and abolishes the contribution of VEGF-D to tumour growth and spread. Further, VEGF-D processing mutants lacking the N- or the C-terminal propeptide displayed distinct effects on tumoural angiogenesis, lymphangiogenesis and tumour growth, however, did not promote metastasis to lymph nodes. This data demonstrates that proteolytic processing of VEGF-D is essential for this protein to enhance angiogenesis and lymphangiogenesis in cancer, and provides a possible strategy by which therapeutics can be targeted to block processing in tumours expressing VEGF-D, in order to restrict the growth and spread of cancer