In Drosophila, eye development is coordinately regulated by the Retinal Determination Gene Network (RDGN), mainly consisting of dachshund (dac), eyes absent (eya), and sin oculis (so). The mammalian homologues are Dach, Eya, and Six, respectively. RDGN, although best known for its role in eye specification, is essential for the development of many organs including retinal, kidney, brain, and limb in both flies and mammals. Molecular mechanism studies support a model that Six functions as a DNA-binding factor and Dach/Eya are transcription co-factors. Eya was first characterized as a nuclear factor, interacting with Six and Dach to regulate gene expression in a context-dependent manner. Eya encodes intrinsic protein tyrosine phosphatase activity. Recently, altered expression of the DACH1/Eya/Six complex has been observed in human tumors. EYA and SIX genes are up-regulated and DACH genes are down-regulated. EYA expression was enhanced in breast, ovary and malignant peripheral nerve sheath tumors. To elucidate the function of Eya in human breast cancer and identify the down-stream targets, we established several human breast cancer cell lines (SKBr3, MDA-MB-453 and BT474) stably expressing Eya1 or phosphatase-defective mutant Eya1D327A. In all three cell lines, expression of Eya1 promoted contact-dependent and contact-independent (soft agar assay) growth. MTT assays and growth curves (cell counting) demonstrated enhanced cellular proliferation upon expression of Eya1. In contrast, cells expressing phosphatase-defective mutant Eya1D327A repressed growth were observed. Western blot analysis demonstrated increased abundance of cell Cyclin D1 in EYA1 expressing cells. Quantitative RT-PCR demonstrated about 3-fold increase of cyclin D1 mRNA abundance in MDA-MB453 cells stably expressing wild type Eya1, but not phosphatase-defective EYA1 mutant. EYA1 induced the human cyclin D1 promoter activity evaluated by luciferase assay and the EYA1 phosphatase activity was required. Immunoprecipitation studies demonstrated association of Eya1 with endogenous Six1. As EYA does not have DNA binding ability and SIX1 is known to binds to the cyclin D1 promoter, we hypothesized model in which EYA1 up-regulates SIX1, and thus a EYA1/SIX1 complex activates Cyclin D1. Lentiviral shRNAs targeting human SIX1 or cyclin D1 were transduced into EYA1 over-expressing cells respectively. The growth curve, MTT assays and clone formation results indicated the pro-proliferate effect of EYA1 was abolished by knock down the expression of either cyclin D1 or Six1. Cyclin D1 abundance was reduced by shSIX1. Taken together, our study demonstrated that cyclin D1 is a key target of EYA1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2942. doi:10.1158/1538-7445.AM2011-2942