Expression of delta-like ligand 4 (Dll4) and markers of hypoxia in colon cancer

Colorectal cancer is the second most common cause of cancer deaths worldwide, with an estimated 1 023 152 new cases and 528 978 related deaths in 2002(Parkin et al, 2005). The early growth of colorectal tumours requires angiogenesis (Goodlad et al, 2006; Korsisaari et al, 2007), the consequence of increased expression of pro-angiogenic factors (e.g., vascular endothelial growth factor-A (VEGF-A); Ferrara et al, 1991; Kim et al, 1993; Jubb et al, 2003; Korsisaari et al, 2007). The expression of VEGF in cancer is controlled by both oncogenic signalling (such as Wnt-signalling in colorectal cancer; Zhang et al, 2001) and hypoxia (Mizukami et al, 2004). Although there is redundancy among pro-angiogenic factors in advanced cancer (Relf et al, 1997; Hanrahan et al, 2003), many early cancers (Hanrahan et al, 2003; Jubb et al, 2003) and in vivo cancer models (Bergers et al, 1999; Hanrahan et al, 2003; Joyce et al, 2003; Goodlad et al, 2006; Korsisaari et al, 2007) are VEGF dependent. This observation has been exploited by the addition of an anti-VEGF monoclonal antibody (bevacizumab) to first-line chemotherapy in metastatic colorectal cancer, which prolonged the median overall survival (from 15.6 to 20.3 months, P=0.001; Hurwitz et al, 2004). Nevertheless, all patients in this trial eventually progressed after 2 years (Jubb et al, 2006b), and there are no valid predictors of the survival benefit afforded by bevacizumab in colorectal cancer (Ince et al, 2005; Jubb et al, 2006a; Grothey et al, 2008a, 2008b). Additional therapeutic agents that disrupt functional tumour angiogenesis have been developed to target tumours that are inherently resistant to anti-VEGF therapy or become resistant during the course of therapy (Noguera-Troise et al, 2006; Ridgway et al, 2006; Pan et al, 2007; Caunt et al, 2008). Delta-like Ligand 4 (Dll4) is a ligand for Notch 1, 3 and 4 proteins, which is expressed by endothelial cells (Thurston et al, 2007; Indraccolo et al, 2009) and may be induced by VEGF and hypoxia, through hypoxia-inducible factor (HIF)-1α (Patel et al, 2005). In the retina, the interaction between Dll4 and Notch regulates endothelial sprouting and branching to form functional vascular beds (Hellstrom et al, 2007). Disruption of Dll4 signalling by overexpression or inhibition of Dll4 may impair tumour angiogenesis (Noguera-Troise et al, 2006; Ridgway et al, 2006), and blockade of Dll4–Notch signalling results in an increased density of non-functional vasculature and is associated with a reduction in the growth of human xenografts (Noguera-Troise et al, 2006; Ridgway et al, 2006). Indeed, certain xenografts that are resistant to anti-VEGF therapy are reported to be sensitive to anti-Dll4 (Noguera-Troise et al, 2006; Ridgway et al, 2006; Li et al, 2007), and combination treatment with anti-VEGF and anti-Dll4 has additive effects on tumour growth (Ridgway et al, 2006). In addition, human umbilical vein endothelial cells transfected with Dll4 downregulate the VEGF receptor 2 and co-receptor neuropilin-1, and show reduced proliferative and migratory responses to VEGF (Williams et al, 2006). Together, these data suggest that Dll4 may have a role in mediating resistance to anti-VEGF therapies. The characterisation of Dll4 protein expression in human cancer is important for the rational design of clinical trials to test the safety and activity of anti-Dll4 therapy. Moreover, defining the context of Dll4 expression, in terms of known markers of hypoxia and angiogenesis, may identify subgroups of tumours with distinct clinical behaviour and response to treatment. The aims of this study were to characterise the in situ expression of Dll4 in colon cancer, to assess the association between Dll4 and established markers of hypoxia and angiogenesis and to determine the prognostic significance of these markers.