Cyr61, a member of the CCN family of genes, is an angiogenic factor. We have shown that it is overexpressed in invasive and metastatic human breast cancer cells and tissues. Here, we investigated whether Cyr61 is necessary and/or sufficient to bypass the 'normal' estrogen (E2) requirements for breast cancer cell growth. Our results demonstrate that Cyr61 is sufficient to induce MCF-7 cells to grow in the absence of E2. Cyr61-transfected MCF-7 cells (MCF-7/Cyr61) became E2-independent but still E2-responsive. On the other hand, MCF-7 cells transfected with the vector DNA (MCF-7/V) remain E2-dependent. MCF-7/Cyr61 cells acquire an antiestrogen-resistant phenotype, one of the most common clinical occurrences during breast cancer progression. MCF-7/Cyr61 cells are anchorageindependent and capable of forming Matrigel outgrowth patterns in the absence of E2. ERa expression in MCF-7/Cyr61 cells is decreased although still functional. Moreover, MCF-7/Cyr61 cells are tumorigenic in ovariectomized athymic nude mice. The tumors resemble human invasive carcinomas with increased vascularization and overexpression of vascular endothelial growth factor (VEGF). Our results demonstrate that Cyr61 is a tumor-promoting factor and a key regulator of breast cancer progression. This study provides evidence that Cyr61 is sufficient to induce E2-independence and antiestrogen-resistance, and to promote invasiveness in vitro, and to induce tumorigenesis in vivo, all of which are characteristics of an aggressive breast cancer phenotype.

We have shown that Cyr61, an angiogenic regulator, is overexpressed in invasive and metastatic human breast cancer cells and tumor biopsies. We have further demonstrated that Cyr61 promotes acquisition of estrogen-independence and anti-estrogen resistance in vivo in breast cancer cells. Moreover, we have demonstrated that Cyr61 induces tumor formation and tumor vascularization in vivo, events mediated through the activation of the MAPK and the Akt signaling pathways. Here we investigate how Cyr61 expression is regulated in both estrogen receptor (ER)-positive and ER-negative breast cancer cells. We demonstrate that Cyr61 mRNA and protein expression is inducible by estrogen and anti-estrogens in ER-positive breast cancer cells. We show that a labile protein as well as a negative regulator might be involved in Cyr61 expression in estrogen-dependent breast cancer cells. Other important regulators of Cyr61 expression in breast cancer cells that we found are the phorbol ester TPA, vitamin D, and retinoic acid. TPA causes positive regulation of Cyr61 expression in ER-positive MCF-7 cells. Vitamin D induces a transient stimulatory eect on Cyr61 gene expression. Lastly, retinoic acid has a negative eect on Cyr61 expression and downregulates its expression in MCF-7 cells. Interestingly, most of these eects are not seen in aggressive breast cancer cells that do not express ER and express high levels of Cyr61, such as the MDA-MB-231 cells. Our results are in agreement with our knowledge that Cyr61 promotes tumor growth, and that tumor-promoting agents have a positive impact on cells that express low levels of Cyr61, such as the ER-positive breast cancer cells; however, these agents have no signi®cant eect on cells that express high levels of Cyr61. Our ®ndings suggest an association between increased Cyr61 expression and an aggressive phenotype of breast cancer cells. Oncogene (2002) 21, 964 ± 973.