ANGIOPOIETIN-2 OVEREXPRESSION PROMOTES HEMATOGENOUS METASTASIS IN BREAST CANCER

Angiogenesis supports tumor growth and facilitates metastasis, the leading cause of patient mortality in breast and other types of solid tumors. Angiopoietin-2 (Ang-2) is an angiogenic factor whose overexpression is associated with increased tumor vascularity, metastasis, and decreased patient survival. We assessed the effects of Ang-2 on breast tumor vasculature by comparing vascular morphology and metastasis of orthotopically implanted metastatic breast carcinoma line MDA-MB-231 that either lacked or overexpressed Ang-2. Methods: Luciferase-tagged MDA-MB-231 breast carcinoma cells designated hAng2 were engineered to overexpress human Ang-2. The control line expressed hAng-2 in reverse orientation. Stable production of hAng-2 was confirmed by RT-PCR, qRT-PCR, Western blot, and ELISA. Functionality of recombinant hAng-2 was assessed by migration and proliferation assays. MDA-MB-231 tumors, hAng2 and control, were orthotopically implanted into female Nu/Nu and SCID mice. Metastasis to lymph node and lung were determined by measuring luciferase activity in tissue extracts. Tumor blood vessel morphology was analyzed by immunohistochemistry (IHC) using antibodies against MECA-32, smooth muscle actin (SMA-alpha), VEGFR-3 and Notch-1. Results: MDA-MB-231 hAng2 lines were stably generated to produce 0-370 ng/ml of hAng-2 while expression in control line was undetectable. Tumor-produced hAng-2 was functional as demonstrated by a dose-dependent induction of migration of lymphatic endothelial cells and mouse mesenchymal stem cells with a maximum increase of 3-fold. Overexpression of Ang-2 had no significant effect on proliferation of cultured MDA-MB-231 cells, growth rate of hAng2 tumors or blood vessel density. In contrast, we detected substantial differences in vascular morphology of Ang-2 overexpressing tumors including 1.7-fold increase in blood vascular area, 2.8-fold increase in number of vessels with open lumen, and 6-fold increase in lumens\u27 cross-sectional area. Blood vascular pericyte coverage shown by SMA-α staining decreased (p\u3e0.001) in hAng2 tumors, demonstrating vessel destabilization. Blood vascular invasion by tumor cells and pulmonary metastasis increased in Ang-2 overexpressing tumors by 500% and 1100%, as compared with control tumors. Blood vessels in hAng2 tumors, but not lymphatic vessels, displayed significantly upregulated Notch-1 and VEGFR-3 expression amplified by a 2.2-fold. Conclusions: These data suggest that Ang-2 increases metastasis because of suppression of pericytes\u27 recruitment that leads to destabilization of the tumor vessels, which facilitates the entry of tumor cells into the vessels and increases metastatic spread. These effects are associated with up-regulation of Notch-1 and VEGFR-3 on tumor vasculature suggesting that signaling of these proteins underlie the morphologic changes in Ang-2 overexpressing tumors. 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 that Ang-2 plays a key role in hematogenous metastasis and suggests that Ang-2 might represent novel a target for inhibition of breast cancer metastasis