VEGF contributes to mammary tumor growth in transgenic mice through paracrine and autocrine mechanisms.

Vascular endothelial growth factor (VEGF) has been identified as a vascular permeability factor, angiogenic cytokine, and a survival factor. To address its role in mammary carcinogenesis, we used transgenic mice with human VEGF(165) targeted to mammary epithelial cells under the control of the mouse mammary tumor virus (MMTV) promoter. Metastatic mammary carcinomas were induced by mating the MMTV-VEGF mice with MMTV-polyoma virus middle T-antigen (MT) mice to generate VEGF/MT mice. Tumor latency was decreased in the VEGF/MT mice, which developed mammary carcinomas with increased vasodilatation at 4 weeks of age. There was increased incidence, multiplicity, and weight of the mammary tumors in 6- and 8-week-old VEGF/MT mice, compared to their MT-only littermates. Macro- and microscopic lung metastases were detected in the VEGF/MT mice but not the MT mice at 6 and 8 weeks of age. Enhanced tumor growth was attributed to increased microvascular density (MVD), as well as increased tumor cell proliferation and survival. Angiogenesis array analysis showed that 24 of 25 differentially expressed genes were upregulated in the VEGF/MT tumors. In vitro studies revealed increased proliferative activity and upregulation of Flk-1 in the VEGF/MT tumor cells, compared with the MT-only tumor cells. Moreover, there was decreased proliferative activity with downregulation of Flk-1 in tumor cells isolated from conditional knockout (VEGF(-/-)) MT-induced mammary carcinomas. The slow growing VEGF(-/-) tumor cells were accumulated in the G(1)/G(0) phase of the cell cycle and this was associated with stimulation of p16(ink4a) and p21(WAF1). Similarly, p16(ink4a) was stimulated in VEGF(lox/lox)/MT mammary tumor cells following Adeno-cre-mediated VEGF gene inactivation. Collectively, the data from these transgenic models indicate that VEGF contributes to mammary tumor growth through increased neovascularization, as well as autocrine stimulation of growth and inhibition of apoptosis.

Long-term exposure to elevated levels of circulating TIMP-1 but not mammary TIMP-1 suppresses growth of mammary carcinomas in transgenic mice.

Tissue inhibitor of metalloproteinases-1 (TIMP-1) regulates matrix metalloproteinase activity, acts as a growth stimulator and inhibits apoptosis. We developed transgenic mice to evaluate the relevance of circulating versus mammary TIMP-1 in mammary carcinogenesis. The transgene was placed under the control of the albumin (Alb) promoter for the production of large amounts of TIMP-1 in the liver and release into the systemic circulation to achieve chronically elevated blood levels. The initial 7,12-dimethylbenz[a]anthracene (DMBA) mammary carcinogenesis study showed greatly decreased tumor incidence in heterozygous Alb-TIMP-1 mice (25%), compared with their wild-type (wt) littermates (83.3%). Metastatic mammary carcinomas were induced in the Alb-TIMP-1 mice through breeding with mice expressing the polyomavirus Middle T antigen (MT) under the control of the mouse mammary tumor virus-long terminal repeat (MMTV-LTR). Both the mammary tumor burden and the incidence of lung metastases were lower in the Alb-TIMP-1/MMTV-MT mice than their MMTV-MT littermates. Analysis of the Alb-TIMP-1/MMTV-MT tumors showed evidence of decreased proliferative activity and inhibition of apoptosis, whereas microvascular density was not affected. Transgenic expression of TIMP-1 in mammary epithelial cells was accomplished by using MMTV-LTR. In contrast to the Alb-TIMP-1 mice, there was insignificant difference in the growth of both DMBA- and MT-induced mammary tumors between heterozygous MMTV-TIMP-1 mice and their wt littermates. The MT-induced mammary tumors of the MMTV-TIMP-1 mice were separated into 'low' and 'high' TIMP-1 expressing groups. The 'high' TIMP-1 expressing tumors exhibited significantly higher proliferative activity than the tumors of the MMTV-MT only mice, whereas the number of apoptotic cells and microvascular density were not different. The findings of this study show that circulating TIMP-1, but not mammary-derived TIMP-1, has growth suppressive effects on DMBA and MT-induced mammary carcinomas.

Tumor growth enhancing effects of vascular endothelial growth factor are associated with increased nitric oxide synthase activity and inhibition of apoptosis in human breast carcinoma xenografts.

Previously, we demonstrated the significance of vascular endothelial growth factor (VEGF) in promoting the growth of tetracycline-regulated human VEGF165 retroviral vector transduced T47-D breast carcinoma cells, particularly at the early stages of tumor development (Cancer Res. 57 (1997) 3924). Here, we showed histologically that the VEGF overexpressing (VEGF (+)) T47-D cells formed a distinct tumor nodule at day 11, while control cells showed no evidence of replication. The VEGF (+) tumors contained large avascular cavities at days 11 and 21, which were replaced by basement membrane-lined channels at day 30. The number of proliferating tumor cells was not significantly different between the VEGF (+) and control tumors, but the number of apoptotic cells was significantly decreased in the VEGF (+) tumors. Increased nitric oxide synthase (NOS) activity was also observed in the VEGF (+) tumors. These findings indicate that VEGF contributes to tumor growth through inhibition of apoptosis and increased NOS activity, which may be critical during pre-vascular stages of tumor development.