Abstract 3564: PlGF/VEGFR-1-dependent activation of the Dll4-Notch4/Ephrin B2 cascade contributes to liver vessel anomalies in hepatocellular carcinoma
Authors: Tijeras-Raballand Annemilai , Contreres Jean-Olivier , Hainaud-Hakim Patricia , Le Hénaff Carole , Pocard Marc , Dupuy Evelyne , de Gramont Armand 
Published: August 2015

Background: Hepatocellular carcinoma (HCC), is characterized by severe vessel anomalies with an intense arterial blood supply, and acquisition of a basal membrane rich in laminin by sinusoids. A role of the VEGF-A/Dll4-Notch4/ephrin B2 cascade in tumor vessel anomalies has been demonstrated, raising the question of the nature of the VEGF receptor pathway implicated in triggering this cascade. This study was undertaken to determinate the role of PlGF and its receptor VEGFR-1 in the development of vascular anomalies in HCC and to evaluate the role of the downstream Notch4 pathway.

Material and Methods: In vitro, we used primary culture of HUVECs, isolated in our laboratory from human umbilical cords. HUVECs were cultured in EBM2 medium, with 20% of FBS and 2ng/ml of FGF-2. The expression of the active form of Notch4 and Dll4 in HUVECs were assessed by Western Blot. In vivo, the differential expression of VEGF-A, PlGF and their receptors were evaluated by qRT-PCR and immunostaining in transgenic mice developing stage-defined HCC.

Results: In vitro, to determine which growth factor/receptor combination was predominant in activating the Notch4 pathway, we compared the effects of VEGF-A, VEGF-E and PlGF on Dll4 expression and Notch4 activation in HUVECs. Treatment with all 3 growth factors increased Dll4 and active Notch4 expressions whose levels were significantly greater in PlGF than VEGF-A and VEGF-E treated HUVECs. Moreover, downregulation of VEGFR-1 by si RNA, but not VEGFR-2, abrogated the effects of VEGF-A and PlGF stimulation on Dll4 expression and Notch4 activation. PlGF silencing significantly reduced Dll4 dependent expression and Notch4 activation in response to VEGF-E and VEGF-A. To confirm in vivo the prevalence of the PlGF/VEGFR-1 pathway in induction of vessel anomalies, we evaluated the differential expression of PlGF, VEGF-A, VEGFR-1 and VEGFR-2 in our transgenic HCC model at different stages. VEGFR-1 expression is increased with HCC progression and restricted in macrophages and sinusoidal endothelial cells. Moreover, PlGF levels were maximal at the stage that coincides with the beginning of liver vessels anomalies in HCC. Silencing PlGF or blocking Notch4 activation, using a γ-secretase inhibitor (DAPT), in vivo delayed tumor growth, reduced arterial vessel length, and sinusoids anomalies with a decrease in Dll4 and active Notch4 expression levels without affecting microvascular density or normal liver.

Conclusions: Together, these data suggest that the activation of PlGF-VEGFR-1/ Dll4- Notch4/ephrin B2 cascades played a role in vessel remodeling and tumor growth. Inhibitor of γ-secretase or silencing PlGF in vivo reduced activation of Notch4, prevent tumor vessels remodelling and arterialization, leading to a delay in tumor growth. These results would argue that targeting PlGF or active Notch4 may be valuable new anti-angiogenic strategies in the treatment of HCC.

Citation Format: Annemilai Tijeras-Raballand, Jean-Olivier Contreres, Patricia Hainaud-Hakim, Carole Le Hénaff, Marc Pocard, Evelyne Dupuy, Armand de Gramont. PlGF/VEGFR-1-dependent activation of the Dll4-Notch4/Ephrin B2 cascade contributes to liver vessel anomalies in hepatocellular carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3564. doi:10.1158/1538-7445.AM2015-3564