Distinct role for the α_vβ₃and the α₆β₁integrin binding sites of Cyr61 in the regulation of breast cancer cell growth and hormone response

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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 α_vβ₃ 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 α_vβ₃. Here, we investigated whether expression of CYR61 mutants with substitutions at the α_vβ₃ (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 α_vβ₃ 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.