Increased levels of vascular endothelial growth factor (VEGF) are associated with a poor response of breast cancer to anti-hormone treatment. Although VEGF is regarded as an endothelial-specific growth factor, recent reports have shown that VEGF can promote proliferation of other cell types, including breast tumor cells. We have characterized the proliferative effects of VEGF in breast cancer cell lines that are commonly used for understanding the role of estrogens, progestins, and anti-hormones on tumor growth. Since steroid hormones can increase the level of VEGF in certain breast cancer cells, we evaluated the effects of exogenous VEGF on the growthsuppressive effects of anti-estrogen (ICI 182,780) and RU-486 (anti-progestin mifepristone) in human breast cancer cells. VEGF 165 and VEGF 121 increased the proliferation of tumor cell lines that expressed VEGFR-2 (VEGF receptor 2) (flk/kdr) via the extracellular signal-regulated kinase/mitogen activated protein kinase (ERK/MAPK) pathway. Furthermore, VEGF induced the expression of the anti-apoptotic protein Bcl-2 and blocked down-regulation of Bcl-2 by ICI 182,780 and induced Bcl-2 in BT-474 and T47-D cells even in the presence of RU-486. Increased Bcl-2 levels in response to VEGF were associated with increased proliferation and survival of tumor cells even in the presence of anti-hormones. These results suggest that VEGF stimulates proliferation of VEGFR2-positive tumor cells, promotes survival via the expression and activity of Bcl-2 and overrides the growth-suppressive effects of anti-hormones. This represents a potential explanation for anti-hormone resistance and tumor progression in clinical samples. Thus, it may be useful to use combined modality treatment involving anti-hormones and anti-angiogenic agents to treat breast cancers that express elevated levels of VEGF.

Recent clinical trials indicate that synthetic progestins may stimulate progression of breast cancer in postmenopausal women, a result that is consistent with studies in chemicallyinduced breast cancer models in rodents. However, progestin-

Angiogenesis, the formation of new blood vessels, is essential for tumor expansion, and vascular endothelial growth factor (VEGF) is one of the most potent angiogenic growth factors known. We have previously shown that natural and synthetic progestins, including those used in hormone replacement therapy and oral contraception, induce the synthesis and secretion of VEGF in a subset of human breast cancer cells in a progesterone receptor-dependent manner. We now report that conditioned medium from progestin-treated breast tumor cells can induce the proliferation of endothelial cells in a paracrine manner and induce the proliferation of tumor epithelial cells in a paracrine and an autocrine manner. The use of an anti-VEGF antibody and SU-1498, an inhibitor of VEGF receptor-2 (VEGFR-2 or flk/kdr) tyrosine kinase activity, demonstrated that these effects involve interactions between VEGF and VEGFR-2. Also, blockage of progestin-induced VEGF by the antiprogestin RU-486 (mifepristone) eliminated VEGF-induced proliferative effects. The ability of VEGF to increase the proliferation of endothelial cells and tumor cells, including those that do not release VEGF in response to progestins, suggests that these effects are mediated by amplification of the progestin signal, which culminates in angiogenesis and tumor growth. These novel findings suggest that targeting the release of VEGF from tumor epithelial cells as well as blocking interactions between VEGF and VEGFR-2 on both endothelial and tumor epithelial cells may facilitate the development of new antiangiogenic therapies for progestin-dependent breast tumors. Furthermore, these data indicate that it would be useful to develop selective progesterone receptor modulators that prevent the release of angiogenic growth factors from breast cancer cells.

Complement 1q-Binding Protein (C1qbp) is a mitochondrial protein reported to be upregulated in cancer. However, whether C1qbp plays a tumor suppressive or tumorigenic role in the progression of cancer is controversial. Moreover, the exact effects of C1qbp on cell proliferation, migration, and death/survival have not been definitely proven. To this end, we comprehensively examined the effects of C1qbp on mitochondrial-dependent cell death, proliferation, and migration in both normal and breast cancer cells using genetic gain- and loss-of-function approaches. In normal fibroblasts, overexpression of C1qbp protected the cells against staurosporine-induce apoptosis, increased proliferation, decreased cellular ATP, and increased cell migration in a wound-healing assay. In contrast, the opposite effects were observed in fibroblasts depleted of C1qbp by RNA interference. C1qbp expression was found to be markedly elevated in 4 different human breast cancer cell lines as well as in ductal and adenocarcinoma tumors from breast cancer patients. Stable knockdown of C1qbp by shRNA in the aggressive MDA-MB-231 breast cancer cell line greatly reduced cell proliferation, increased ATP levels, and decreased cell migration compared to control shRNA-transfected cells. Moreover, C1qbp knockdown elicited a significant increase in doxorubicin-induced apoptosis in the MDA-MB-231 cells. Finally, C1qbp upregulation was not restricted to breast cancer cells and tumors, as levels of C1qbp were also found to be significantly elevated in both human lung and colon cancer cell lines and carcinomas. Together, these results establish a pro-tumor, rather than anti-tumor, role for C1qbp, and indicate that C1qbp could serve as a molecular target for cancer therapeutics.

MCF-7 cells growing in culture were used to study the mechanism of the antiproliferative activity of the antiprogestin mifepristone, as compared with the antiestrogen 4-hydroxytamoxifen or the combination of both. These steroid antagonists induced a significant time- and dose-dependent cell growth inhibition (cytotoxicity). This inhibition of cell survival was associated with a significant increase in DNA fragmentation (apoptosis), downregulation of bcl2, and induction of TGFbeta1 protein. Abrogation of the mifepristone- and/or 4-hydroxytamoxifen-induced cytotoxicity by TGFbeta1 neutralizing antibody confirms the correlation between induction of active TGFbeta1 and subsequent cell death. The effect of a combination of mifepristone and 4-hydroxytamoxifen on cell growth inhibition, on the increase in DNA fragmentation, bcl2 downregulation, and induction of TGFbeta1 protein was additive and significantly different (P < 0.05) from the effect of monotherapy. A translocation of protein kinase C (PKC) activity from the soluble to the particulate and/or nuclear fraction appeared to be also additive in cells treated with a combination of both 4-hydroxytamoxifen and mifepristone. These results suggest that the mechanism of the additive antiproliferative activity of mifepristone and tamoxifen could be explained at least in part by an additive induction of apoptosis in both estrogen and progesterone receptor positive MCF-7 breast cancer cells. A bcl2 downregulation, the PKC transduction pathway, and TGFbeta1 expression seem to be involved in this additive mechanism of action. Our data further suggest that a combination of an antiprogestin with tamoxifen may be more effective than tamoxifen monotherapy in the management of human breast cancer.