Cyr61 promotes breast tumorigenesis and cancer progression

ª 2002 Nature Publishing Group Oncogene (2002) 21, 8178 – 8185 All rights reserved 0950 – 9232/02 $25.00 www.nature.com/onc Cyr61 promotes breast tumorigenesis and cancer progression Miaw-Sheue Tsai1, Daphne F Bogart1,2, Jessica M Castañeda1,3, Patricia Li1 and Ruth Lupu*,1,4,5 1 Ernest Orlando Lawrence Berkeley National Laboratory, University of California, One Cyclotron Road, Berkeley, California, CA 94720, USA 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 MCF7/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. Oncogene (2002) 21, 8178 – 8185. doi:10.1038/sj.onc. 1205682 Keywords: Cyr61; invasiveness; estrogen-dependence; antiestrogen-resistance; breast cancer *Correspondence: R Lupu; E-mail: Current addresses: 2Bayer Corporation, 800 Dwight Way, Berkeley, California, CA 94710, USA; 3Microgenics Corporation, 46360 Fremont Blvd, Fremont, California, CA 94538, USA; 4Evanston Northwestern Healthcare Research Institute, 1001 University Place, Evanston, IL 60201, USA; 5Department of Medicine, Northwestern University Medical School, 1001 University Place, Evanston, IL 60201, USA Received 21 January 2002; revised 15 May 2002; accepted 20 May 2002 About 60% of human breast carcinomas expressing estrogen receptor (ER), are dependent upon estrogen (E2) for growth, and thus respond to treatment with an ER antagonist, tamoxifen (Tam). Many breast carcinomas, however, become less sensitive over time to estrogen, and thus more resistant to the endocrine treatment, developing into more aggressive tumors. Aggressiveness of breast cancer cells is commonly attributed to the ability of the cells to overcome E2 requirements for growth, and in most cases to acquire antiestrogen-resistance. However, the mechanism by which breast cancer progresses from an E2-dependent and antiestrogen-responsive phenotype to an E2independent and antiestrogen-resistant phenotype has not yet been determined. We have shown previously that expression of heregulin (HRG), a growth factor that activates the erbB-2/3/4 receptor signaling pathways, is closely associated with an invasive breast cancer phenotype (Cardillo et al., 1995). We have further demonstrated that HRG induces breast cancer progression, as determined by the loss of ER function and E2 response, tumorigenicity, invasion, and metastasis (Tang et al., 1996; Lupu et al., 1995, 1996; manuscript submitted for publication). We have hypothesized that HRG induces activation of the erbB signaling pathways, leading to regulation of downstream genes that control cancer cell growth and tumor progression. We thus isolated and identified Cyr61, an angiogenic factor, which is differentially expressed in ER-negative, HRG-positive, invasive, and metastatic breast cancer cells, and in 30% of breast tumor biopsies (Tsai et al., 2000). We showed that Cyr61 is important for HRGmediated chemomigration and invasiveness of breast cancer cells in vitro (Tsai et al., 2000). Cyr61 belongs to the CCN family of angiogenic regulators, which consists of Cyr61, CTFG, Nov, WISP-1, WISP-2, and WISP-3 (Lau and Lam, 1999). Cyr61 is a cysteine-rich, heparin-binding protein that is secreted and associated with the cell surface and the extracellular matrix (ECM) (Yang and Lau, 1991; Kireeva et al., 1997). It has been shown that Cyr61 binds to integrins, such as avb3, aIIbb3 and a6b1 (Kireeva et al., 1996a; Jedsadayanmata et al., 1999; Chen et al., 2000). Cyr61 mediates cell adhesion, stimulates cellular migration, enhances growth factorinduced DNA synthesis in fibroblasts and endothelial cells, and increases chondrogenesis in mesenchymal cells (O’Brien and Lau, 1992; Kireeva et al., 1996b, Cyr61 promotes breast cancer progression M-S Tsai et al 8179 1997; Frazier et al., 1996; Kolesnikova and Lau, 1998). Moreover, Cyr61 stimulates an integrin avb3-dependent chemotaxis of endothelial cells (Babic et al., 1998). Most significantly, expression of Cyr61 enhances neovascularization and tumor formation of human tumor cells in immunodeficient mice (Babic et al., 1998, 1999; Xie et al., 2001). To determine whether expression of Cyr61 is necessary and/or sufficient to promote breast cancer progression, Cyr61 was introduced into the MCF-7 breast cancer cells, which are ER-positive, E2-responsive in vitro, E2-dependent in vivo, and never metastasize in vivo. MCF-7 cells were stably transfected by electroporation with a eukaryotic expression vector, pcDNA3.1/zeocine(7) (Invitrogen), containing the full-length cDNA of the human Cyr61 gene (MCF-7/ Cyr61), or with an empty vector (MCF-7/V) as a negative control. A number of Cyr61- (20 clones) and vector-transfected clones (10 clones) were isolated and characterized for the expression of Cyr61 at both the Figure 1 Expression of Cyr61 in MCF-7/Cyr61 clones. (a) MCF7 cells were stably transfected by electroporation with a eukaryotic expression vector, pcDNA3.1/zeocine(7) (Invitrogen), containing the full-length cDNA of the human Cyr61 gene, or with an empty vector as a negative control. Transfected MCF-7 cells were selected in the presence of antibiotic zeocine (200 mg/ml) for 2 weeks. Individual vector transfectants (MCF-7/V, 10 clones) and Cyr61 transfectants (MCF-7/Cyr61, 20 clones) were isolated and grown. Total RNA was isolated from subconfluent MCF-7/ V2 and MCF-7/Cyr61 cells, and 30 mg of RNA was analysed for Cyr61 mRNA expression by RNAse protection assay as previously described (Tsai et al., 2000). The GAPDH probe was used as an internal control for RNA loading. Representative vector (V2) and Cyr61 clones (C6, C19, and C20) w (...truncated)