Vascular endothelial growth factor (VEGF) and Delta-like 4 ligand (DLL4) are the only genes whose haploinsufficiency results in vascular abnormalities. Although many common pathways are up-regulated in both vascular development and tumor angiogenesis and in vascular remodeling, the role of the Delta/Notch pathway has not been clearly defined in tumor angiogenesis. In this study, we assessed the expression of DLL4, Notch4, and ephrin B2 in transgenic mice developing hepatocarcinoma characterized by a strong remodeling of the tumor sinusoids. We also investigated the role of VEGF in the expression and biological functions of these molecules on human venous endothelial cells. In transgenic livers, we showed that DLL4, active Notch4, and ephrin B2 were gradually up-regulated within the hepatocarcinoma progression and expressed on tumor sinusoidal endothelial cells. In venous endothelial cells, we showed that VEGF up-regulates DLL4 and presenilin, and increased the activation of Notch4, leading to an up-regulation of ephrin B2 with a downregulation of Eph B4. We also showed that the activation of Notch4 is required for VEGF-induced up-regulation of ephrin B2 and the differentiation of human venous endothelial cells in vitro. Accordingly, the disruption of Notch4 signaling by pharmacologic inhibition of presenilin or addition of soluble DLL4 inhibited the effect of VEGF on human venous endothelial cell migration and differentiation. Our study strongly suggests that a coordinated activation of DDL4/ Notch4 and ephrin B2 pathways downstream of VEGF plays a key role in the abnormal remodeling of tumor vessels. (Cancer Res 2006; 66(17): 8501-10)

IntroductionHuman T-cell lymphotropic virus type I (HTLV-I) is the etiologic agent of an aggressive and fatal T-cell malignancy of activated CD4 ϩ CD45RO ϩ T lymphocytes termed adult T-cell leukemia/ lymphoma (ATL). 1,2 The mechanisms of leukemogenesis are not yet fully understood. Infection during infancy and a long clinical latency period of 20 to 30 years appear to be critical factors associated with the development of ATL. During this period, clonal expansion of HTLV-I-bearing T cells occurs, and, following a model of multistep oncogenesis, the accumulation of critical somatic mutations may contribute to the development of ATL. Viral protein expression from early infection to ATL may play a major role during all stages of the disease development. 3 The HTLV-I Tax protein is a 40-kDa transcriptional transactivator of the HTLV-I gene via its interaction with activation transcription factor (ATF)/CCAAT-enhancer binding protein (CREB) proteins and the transcriptional coactivators CREB binding protein (CBP) and p300. 4,5 Tax is also capable of increasing expression of other cellular genes by positively regulating nuclear factorB (NF-B) activity. 3 There is strong evidence that Tax may also play a critical role in the cellular transformation of various in vitro models, including T cells, and is capable of inducing tumors in transgenic mice. [6][7][8] In these models, Tax induction of transformation is also associated with cellular gene expression modulation via the NF-B and/or ATF/CREB pathways. 9 In ATL cells, activated protein-1 (AP-1) activity is constitutively activated 10,11 and may play a critical role in cell proliferation and transformation. AP-1 is a transcription factor complex composed of members of Fos (c-fos, FosB, Fra-1, and Fra-2) and Jun (c-Jun, JunB, and JunD) families that play a major role in the positive regulation of proliferation and activation of T-cell and cytokine production. 12,13 In nonstimulated normal T cells, the basal level of AP-1 proteins is low, but T-cell activation results in rapid induction of jun and fos genes. 14 AP-1 activity is also regulated at the posttranscriptional level by the activation of c-Jun N-terminal kinase (JNK). 15 JNK phosphorylates c-Jun, thereby increasing its DNA binding activity. 16 this pathway by inducing the expression of various members of the AP-1 family, including c-Jun, and by constitutively activating JNK. 10,13,17,18 Several reports have demonstrated that fresh ATL cells as well as ATL cell lines produce high levels of transforming growth factor ␤1 (TGF-␤1) as a consequence of the activation of AP-1 sites located in the 5Ј regulatory region of the TGF-␤1 gene. 19,20 However, the role of TGF-␤1 production by ATL cells in HTLV-I leukemogenesis remains to be elucidated.TGF-␤1 controls various aspects of cell growth and differentiation by signaling through a heteromeric complex of type I (TGF-␤1-RI) and II (TGF-␤1-RII) serine/threonine kinase transmembrane receptors. TGF-␤1 binds TGF-␤1-RII, resulting in the recruitment and the activation of TGF-␤1...