Abstract Homeobox B9 (HOXB9) is a member of the class I HOX genes with highly conserved 61 amino acid homedomain motif, known to be overexpressed in breast cancer and related to tumor angiogenesis, EMT (Hayashida et al., PNAS 2010), and radio-resistance (Chiba et al., PNAS 2011), being significant prognostic factor in breast cancer patients (Seki et al., Ann Surg Oncol 2012). In this study, we investigated how HOXB9 expression is regulated in breast cancer cells. First, we analyzed the upstream promoter region of HOXB9 gene to determine the important sequence for HOXB9 transcription. The multiple lengths of HOXB9 promoter region from -2898 to +1 were cloned into pGL3 luciferase reporter vector. Luciferase activities in MDA-MB231 cells which overexpress HOXB9 were analyzed and determined that the region from -404 to -392 played the critical role for the transcription of HOXB9 gene. Computer prediction system for the binding ability to critical sequence raised several candidate genes including E2F-1, PAX-5, P53, Sp-I, ETS-1 and NFY-A which are known to be related with cancer progression. It was confirmed that all these genes could induce HOXB9 overexpression, and especially E2F-1 was the strong inducer. Then, E2F-1-induced cell lines from MCF7, MCF10A and MDA-MB231were established and the correlation between E2F-1 and HOXB9 mRNA expression was determined. ChIP assay revealed that E2F-1 could directly bind to the critical sequence of HOXB9 promoter. Electrophoretic Mobility Shift Assay (EMSA) also indicated the binding ability of E2F-1. Finally, the tissue samples of 141 breast cancer patients, who undertook breast cancer surgery as a primary treatment, were stained with E2F-1 and HOXB9 antibodies, and correlation between HOXB9 and E2F-1 expression was confirmed with statistical significance (p<0.001). There are many publications describe the E2F-1 relation to the breast cancer patients prognosis. The context of this research displays the direct dependence of HOXB9 gene's expression through E2F-1, which might be suppressed by specific treatment, for example CDK4/6 inhibitor, and improve breast cancer patients’ survival. To confirm these genes might demonstrate novel factors and/or signaling pathways playing role in breast cancer progression through HOXB9 axis. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-07-06.