Stromal-epithelial interactions drive development and maintain tissue homeostasis through a network of soluble and insoluble factors that operate within a three-dimensional (3D) tissue. Genetic and epigenetic changes in mammary epithelial cells (MECs) cooperate with a modified tissue microenvironment to drive malignant transformation of the breast. We have been studying how altered expression of developmental regulators contributes to breast tumorigenesis. Hox genes play a critical role in tissue development, are frequently lost in tumors, and can regulate integrin and ECM expression. Global expression analysis of matched tumor/normal breast tissue revealed that HoxA9 expression was significantly lower in the tumors. Q-RT-PCR, in situ hybridization and immunohistochemical analysis verified that HoxA9 expression was epithelial specific and significantly reduced in a cohort of primary breast tumors and breast cancer lines. Re-expression of HoxA9, and not HoxA10, in breast cancer cells significantly repressed their tumorigenic phenotype in culture and in vivo coincident with induction of the tumor suppressor BRCA1. In addition, we found that HoxA9 re-expression regulated adhesion by altering integrin expression and adhesion activity resulting in phenotypic reversion of breast tumor cells grown within a 3D reconstituted basement membrane (3D rBM). HoxA9 consensus binding sequences in the BRCA1 promoter were verified in breast cells by mutational studies and ChIP and luciferase analysis. Moreover, exogenous expression of BRCA1 phenocopied and BRCA1-Δexon11b expression and BRCA1 shRNA compromised HoxA9\u27s in vivo and in vitro breast tumor suppressor effects. Because we found that the HoxA9 gene is methylated in breast cancer cells, can repress breast cancer behavior, and is coordinately expressed with BRCA1 in normal and transformed breast tissue but not in endothelial cells, we conclude that HoxA9 is an attractive mammary-specific candidate tumor modifier gene
