This project focuses on the roles of VEGF and VEGF-D in breast cancer development and progression. Transgenic mice with overexpression of VEGF in mammary epithelial cells were crossed with Middle T mice, which develop metastatic mammary carcinomas. Mammary carcinomas emerged earlier in the VEGF overexpressing mice and showed greatly dilated blood vessels, increased vascular density, and upregulation of multiple angiogenesis-associated genes. There was a significant increase in the number of proliferating tumor cells and decreased number of apoptotic tumor cells in the VEGF overexpressing tumors. The most novel finding of this project was the presence and upregulation of the VEGF receptors, Flk-1 and Nrp-1 on the tumor cells besides the endothelial cells within the VEGF overexpressing. Parallel to the in vivo findings, an in vitro study of tumor cells isolated from the carcinomas showed increased growth, upregulation of Flk-1 and Nrp-1, activation of Flk-1, and co-localization of the two receptors in the VEGF overexpressing carcinomas. These findings suggest that VEGF has an important autocrine function in mammary carcinoma cells, in addition to its well established paracrine function as an endothelial mitogen. Cre-Lox mice were used to inactivate the VEGF gene in mammary epithelial cells of virgin and pregnant mice. Conditional knockout of one VEGF allele in virgin mammary glands, using the MMTV promoter, had dramatic developmental effects with severe inhibition of lobulo-alveolar formation and almost complete cessation of milk production. Mammary gland development was much less affected when VEGF was knocked out at the end of pregnancy by the use of the WAP promoter. However, these mammary glands showed focal patches of epithelial cells apoptosis, suggestive of unscheduled involution. This finding suggests that VEGF is a survival factor for nonneoplastic mammary epithelial cells. It will be important to characterize the role of VEGF in the pathogenesis of common benign breast diseases like fibrocystic disease and sclerosing adenosis. Further indication of a role of VEGF as a survival factor came from studies of mammary carcinomas induced by Middle T in the WAP-Cre/VEGF-Lox model. The few VEGF knockout tumors observed so far had a latent period of approximately nine months, compared to eight weeks in the Middle T only mice. Primary outgrowths of tumor cells from these carcinomas showed morphological evidence of senescence in vitro and induction of the senescence markers, p16 and p21. Although preliminary, these findings further support the a vital function of VEGF for growth and survival of mammary carcinoma cells. NRP-1 is a VEGF-165 specific receptor. It uses KDR-Flk-1 as a signaling co-receptor in endothelial cells, and plexin-A1 or L1-CAM in neuronal cells. Based on the finding of NRP-1 upregulation in mammary carcinoma cells in our transgenic model, we decided to examine if NRP_1 and its neuronal co-receptors were expressed present in human breast carcinomas. Immunohistochemistry and confocal microscopy showed overlapping expression of Nrp-1, Plexin-A1, and L1-CAM in infiltrating ductal carcinomas withed co-localization of NRP-1 and Plexin-A1 in tumor cells. Plexin-A1 overexpressing breast carcinoma cells responded to VEGF-165 stimulation in culture by decreased phosphorylation of LIM kinase and its down-stream target, cofilin, which are known to be involved in F-actin rearrangement associated with cell migration. The role of L1-CAM in hypoxia-induced anti-apoptotic effect of VEGF was established, using flow cytometric analysis. This involved phosphorylation of Akt. These studies indicate that the neuronal signaling receptors, Plexin-A1 and L1-CAM, contribute to autocrine VEGF functions involving migration and apoptosis in human breast carcinoma cells. We identified a novel splice variant of NRP-1 from a human lung cDNA library. This variant encodes a soluble form of NRP-1 and was named s10NRP-1 because it possesses unique intron 10 sequences. We generated transgenic mice overexpressing s10NRP-1 in skeletal muscle to obtain high circulating levels of the protein. This mouse model is used to test the hypothesis that circulating s10NRP-1 acts as a tumor suppressor by competing with VEGF binding to the full-length NRP-1. Middle T-induced mammary carcinogenesis studies are ongoing in heterozygous S10NRP-1 mice and their wildtype littermates. VEGF-D is a ligand for the VEGF receptors, KDR/Flk-1 and the lymphatic Flt-4. Immunohistochenical studies localized VEGF-D in vascular and bronchial smooth muscle cells, as well as ductal myoepithelial cells in human breast and lung tissues. VEGF-D was found to stimulate growth of vascular and bronchial smooth muscle cells, which was associated with increased phosphorylation of KDR/Flk-1 and activation of the MAPK pathway. Staining of human breast cancer samples revealed faint VEGF-D positivity in breast cancer cells. Consequently, a study was undertaken to characterize the effects of forced VEGF-D expression on human breast carcinoma cell lines. Data from MCF-7 and MDA-MB-231 lines showed upregulation of Bcl-2 and downregulation of Bax with increased Bcl-2/Bax ratio. When treated with the apoptosis inducers, staurosporin and cycloheximide, increased survival of the VEGF-D overexpressing clonal lines was observed, suggesting that VEGF-D is a survival factor for human breast carcinoma cells. This project focuses on the roles of VEGF and VEGF-D in breast cancer development and progression. Transgenic mice with overexpression of VEGF in mammary epithelial cells were crossed with Middle T mice, which develop metastatic mammary carcinomas. Mammary carcinomas emerged earlier in the VEGF overexpressing mice and showed greatly dilated blood vessels, increased vascular density, and upregulation of multiple angiogenesis-associated genes. There was a significant increase in the number of proliferating tumor cells and decreased number of apoptotic tumor cells in the VEGF overexpressing tumors. The most novel finding of this project was the presence and upregulation of the VEGF receptors, Flk-1 and Nrp-1 on the tumor cells besides the endothelial cells within the VEGF overexpressing. Parallel to the in vivo findings, an in vitro study of tumor cells isolated from the carcinomas showed increased growth, upregulation of Flk-1 and Nrp-1, activation of Flk-1, and co-localization of the two receptors in the VEGF overexpressing carcinomas. These findings suggest that VEGF has an important autocrine function in mammary carcinoma cells, in addition to its well established paracrine function as an endothelial mitogen. Cre-Lox mice were used to inactivate the VEGF gene in mammary epithelial cells of virgin and pregnant mice. Conditional knockout of one VEGF allele in virgin mammary glands, using the MMTV promoter, had dramatic developmental effects with severe inhibition of lobulo-alveolar formation and almost complete cessation of milk production. Mammary gland development was much less affected when VEGF was knocked out at the end of pregnancy by the use of the WAP promoter. However, these mammary glands showed focal patches of epithelial cells apoptosis, suggestive of unscheduled involution. This finding suggests that VEGF is a survival factor for nonneoplastic mammary epithelial cells. It will be important to characterize the role of VEGF in the pathogenesis of common benign breast diseases like fibrocystic disease and sclerosing adenosis. Further indication of a role of VEGF as a survival factor came from studies of mammary carcinomas induced by Middle T in the WAP-Cre/VEGF-Lox model.
| Buck, T B; Hall, A L; Sinha, C C et al. (2000) cis-hydroxyproline stimulates the growth of rat mammary carcinoma cells. In Vivo 14:11-Jul |
| Schoeffner, D J; Thorgeirsson, U P (2000) Susceptibility of nonhuman primates to carcinogens of human relevance. In Vivo 14:149-56 |
| Harris, S R; Panaro, N J; Thorgeirsson, U P (2000) Oxidative stress contributes to the anti-proliferative effects of flavone acetic acid on endothelial cells. Anticancer Res 20:2249-54 |
| Panaro, N J; Popescu, N C; Harris, S R et al. (1999) Flavone acetic acid induces a G2/M cell cycle arrest in mammary carcinoma cells. Br J Cancer 80:1905-11 |
