The pro-angiogenic regulator Cyr61 is overexpressed in invasive breast cancer cells and tumor biopsies. The expression of Cyr61 is sufficient to promote breast cancer cell proliferation, cell survival and chemosensitivity through a αvβ3 integrin-activated ERK1/ERK2 MAPK signaling. Additionally, Cyr61 induces breast cancer cells to bypass the normal estrogen requirements for growth and acquire an estrogen-independent and antiestrogen resistant phenotype. This is apparently mediated via the interaction with the integrin receptor αvβ3. Here, we investigated whether expression of CYR61 mutants with substitutions at the αvβ3 (D125A) or α6β1 (TM) binding domains might impede CYR61’s ability to induce an estrogen (E2)-independent and/or the antiestrogen resistant phenotype in MCF-7 cells. To do so, retroviral infection was used to generate MCF-7 stably cell lines expressing either the CYR61–D125A or the CYR61-TM cDNA variants. Matched controls expressing an empty pBabe vector were also generated. CYR61 expression levels were monitored by immunoblotting. To determine the response of the MCF-7/D125 and MCF-7/TM cells to estrogen and antiestrogen stimuli we performed a series of studies in the presence or absence of E2 and in the presence or absence of the antiestrogens Tamoxifen and ICI 182,782. Our results demonstrates that, while MCF-7/D125A cells anchorage-independent in the absence of E2, similarly to MCF-7/CYR61 cells; MCF-7/TM cells remained E2-dependent forming a number of colonies similar to MCF-7/WT and MCF-7/pBabe cells. These results reveal that CYR61 signaling through α6β1 integrin allows the acquisition of an E2-independent and antiestrogen resistant phenotype in breast cancer cells. Moreover, cell cycle progression analysis of MCF-7/D125A and MCF-7/TM cells showed a decreased proliferative response in comparison to the MCF-7/CYR61 cells as measure by flow cytometric analysis. These findings suggest that, whereas mutations in both, the αvβ3 or the α6β1-binding domain impair the CYR61-induced cell proliferation; the α6β1 binding domain is critical for the CYR61-triggered signaling that results in a hormone-independent phenotype in breast cancer cells. Collectively, these data point toward a differential role for the CYR61 integrin binding domains in the regulation of the cell proliferation and hormone response in breast cancer.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]