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#3041 PR expression is inversely associated with the stage of breast cancer and loss of PR is associated with more aggressive phenotype suggesting that PR has a protective role in established cancer. However, animal models and clinical studies showed that progesterone (PG) and PR is a risk factor for breast cancer development. The mechanism of these distinct actions of PG/PR in breast cancer is not known. PR contains a polyproline domain (PPD), which binds to the SH3 domain of c-Src. PR-SH3 interaction mediates activation of Src and MAPK and PG-mediated cell cycle progression. PR-BΔSH3, which has mutations in PPD, failed to mediate these effects. To access the biological role of PR extra-nuclear signaling mediated by PPD, PR-B, PR-BΔSH3 or vector control were stably expressed in non-transformed mammary epithelial MCF10A cell and in breast cancer cells, MCF-7 and T47DY. Cells were tested for their transformation potential by growth in soft-agar and in 3-D cultures. In MCF10A model, cells expressing PR-BΔSH3 showed an increase in both colony numbers and sizes while cells expressing PR-B showed little growth in soft-agar in the absence of PG and PG strongly inhibited colony formation. In MCF-7 cells, both cells expressing PR-B and PR-BΔSH3 formed colonies in the absence of PG and PG strongly inhibited colony formation in cells expressing PR-B but had no effects in cells expressing PR-BΔSH3. To mimic growth in vivo, cells were grown in 3D cultures. MCF-10A expressing vector control or PR-B formed polarized acini with hollow lumen while cells expressing PR-BΔSH3 formed large and filled acini. Using Ki-67 and E-cadherin immunostaining, cells expressing PR-BΔSH3 showed an increase in cells proliferation and a break down in E-caderin as compared to cells expressing PR-B or vector control. Similar changes in cells expressing PR-BΔSH3 were also found in MCF-7 model. We next determined the influence of PR PPD on cell morphology and motility in T47DY PR- cells. T47DY expressing PR-B were rounded and epithelial-like as compared to cells expressing PR-BΔSH3 or LacZ, which were large, flat and mesenchymal-like. Cells expressing PR-B showed a decrease in cell motility with few focal adhesions and were devoid of actin stress fibers, as compared to cells expressing PR-BΔSH3 and LacZ. Similar changes were also observed in MCF-7 cell series. Thus, PR PPD appears to play a role in epithelial-mesenchymal transition (EMT) of breast cancer cells. Interestingly, effects of PR-B on these cellular functions are independent of ligand, but dependent upon PR PPD. Using DNA microarrays, gene expression of T47DY expressing PR-B and PR-BΔSH3 were compared. Gene ontogeny analysis of genes regulated by PPD in unliganded receptor revealed an enrichment of genes involved in regulation of key cellular processes including cell polarity, motility, EMT, NF-KB, TGF-β, and Wnt signaling. Results from DNA microarrays demonstrate that extra-nuclear signaling of PR through PPD interactions has a profound effect on PR regulated gene in the absence of PG and may plays an important role in mediating differentiating effects of PR. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3041.