In vitro studies on interactions between VEGF and endothelin-1 signaling in human breast cancer cells
Authors: Jeanett Fischgrabe, Martin Gotte, Martin Smollich, Ludwig Kiesel, Pia Wulfing
Journal: Cancer Research (2008)

AACR Annual Meeting-- Apr 12-16, 2008; San Diego, CA

1129 

Background: Endothelin-1 (ET-1) and vascular endothelial growth factor (VEGF) are known to promote growth and survival of vascular endothelial cells in a synergistic fashion. Both peptides have been shown to play crucial roles in tumor vascularization. Angiogenic effects of ET-1 and VEGF are mainly mediated via the G protein-coupled ET receptor A (ETAR) and VEGF receptor 2 (KDR), respectively. In breast cancer, increased expression of ET-1 and VEGF has been shown to be associated with poor prognosis. Moreover, we have previously shown ET-1 expression to correlate with expression of VEGF and micro vessel density in breast tumour tissue samples. Since inhibition of angiogenesis represents a promising therapeutic approach in breast cancer, enlightenment of underlying pathways may contribute to future clinical success of anti-angiogenic targeted therapies. Against this background, we studied potential interactions between the ET-1 and VEGF pathway in breast cancer cells, by stimulating the cells with ET-1, VEGF and the respective inhibitors: the selective ETAR antagonist atrasentan (ABT-627) and the humanized monoclonal VEGF antibody bevacizumab.    Methods: Various human breast cancer cell lines (MCF-7, MDA-MB-231, MDA-MB-468, SKBR-3, BT-474) were incubated with ET-1 (100 nM) +/- ABT-627 (1 µM), VEGF (100 ng/mL) +/- bevacizumab (10µg/mL), respectively, in a time dependent manner (2, 8, and 24 hours). Subsequently, gene expression levels of VEGF, KDR, ET-1, ETAR and further related genes were determined by quantitative real time PCR utilizing the appropriate TaqMan® probe. In cell culture supernatants VEGF and ET-1 secretion was determined by ELISA.    Results: In neither cell line investigated, stimulation with ET-1 was capable of modulating VEGF, KDR and HIF-1α gene expression. Also, there was no impact of VEGF on gene expression of the endothelin axis. Regarding protein secretion, VEGF has been shown to induce ET-1 secretion in SKBR-3 (125%; p ≤ 0.05) and MDA-MB-468 (110%; p ≤ 0.05) breast cancer cells. As expected, in both cell lines this effect was abrogated by bevacizumab. In MDA-MB-468 breast cancer cells ET-1 secretion was reduced to 55% (p ≤ 0.05 compared to control) of vehicle treated cells. ET-1 secretion of BT-474 and MCF-7 breast cancer cells was not affected by VEGF. However, incubation of both cell lines with bevacizumab resulted in decreased ET-1 secretion (BT-474: 52%, p ≤ 0.001; MCF-7: 81%, p ≤ 0.05). Incubation with ET-1+/- ABT-627 did not alter VEGF secretion.    Conclusions: Our studies have shown that - in contrast to other tumour entities - there are only moderate, gene expression-independent interactions of VEGF and the ET-axis in breast cancer cells. Nevertheless or even therefore, simultaneous targeting of both pathways in breast cancer may represent a promising therapeutic approach to achieve synergistic inhibition of angiogenesis and tumor growth in breast cancer patients.