Abstract 2354: Angiopoietin-2 role in breast cancer metastasis

Abstract

Abstract Background: Angiopoietin-2 (Ang-2) is an angiogenic factor whose overexpression is associated with increased tumor vascularity, metastasis, and decreased patient survival. The pro-angiogenic effect of Ang-2 is mediated through activation of the Tie-2 receptor that leads to vessel destabilization and dismantling. The latter is an essential step for both angiogenesis and tumor entry into the lumen. We assessed the effects of Ang-2 on tumor vasculature in orthotopic breast carcinoma model MDA-MB-231 that either lacked or overexpressed this factor. Methods: Luciferase-tagged MDA-MB-231 cells were engineered to overexpress human Ang-2. The control line expressed Ang-2 in reverse orientation. Stable production of Ang-2 was confirmed by RT-PCR, qRT-PCR, Western blot, and ELISA. Ang-2 functionality was assessed by migration and proliferation assays using tumor and endothelial cells. Ang2-overexpressing and control MDA-MB-231 cells were orthotopically implanted into mice. Metastasis to lymph node and lung were determined by measuring luciferase activity in tissue extracts. Tumor blood vessel morphology was analyzed by immunohistochemistry using antibodies against MECA-32, smooth muscle actin (SMA-α), VEGFR-3 and Notch-1. Results: Ang-2-overexpressing tumor line produced 11±0.4 ng/ml of Ang-2 while expression in control line was undetectable. Tumor-produced Ang2 was functional as demonstrated by a dose-dependent migration of lymphatic endothelial cells. Soluble Tie-2 inhibited Ang-2 induced migration by 70%. Ang-2 had not effect on proliferation of MDA-MB-231 cells, tumor growth or blood vessel density. However, Ang-2 overexpressing and control tumors substantially differed in vascular morphology. The differences included 170% increase in blood vascular area, 280% increase in number of vessels with open lumen, and 600% increase in lumens’ cross-sectional area. Pericyte coverage shown by SMA-α staining decreased >10-fold (p>0.001) in Ang-2 tumors, demonstrating vessel destabilization. Tumor vascular invasion and pulmonary metastasis increased by 500% and 1100% in Ang-2 tumors compared with controls. Also, blood vessels in Ang-2 tumors displayed 2.0-2.2 fold upregulated Notch-1 and VEGFR-3 expression. Conclusions: These data suggest that Ang-2 increases metastasis via suppression of pericytes’ recruitment, which destabilizes tumor vessels and facilitates entry of tumor cells into the lumen. The increased expression of Notch-1 and VEGFR-3 on blood vessels in Ang-2 overexpressing tumors suggests that signaling of these proteins underlie the vascular morphologic changes in Ang-2-rich environment. This is consistent with prior data demonstrating up-regulation of VEGFR-3 on tumor blood vessels and association of both proteins with increased metastasis. This study demonstrates a key role for Ang-2 in hematogenous metastasis and suggests that suppression of Ang-2 and VEGFR-3 could inhibit breast cancer metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2354.