ABSTRACTIt is well accepted that intrinsic action of the androgen receptor (AR) within the prostate epithelium drives prostatecancer proliferation and survival. Less appreciated is the fact that AR is also expressed in the stroma surroundingthe epithelium. Stromal-expressed AR acts extrinsically to maintain the differentiated striated basal and luminalepithelium of the normal gland. During prostate cancer development AR expression in the stroma is lost. HowAR is lost from the stroma and how its loss promotes prostate cancer development is unknown. Our objectivesare to define the mechanism that leads to stromal-specific AR loss and determine how AR loss in the stroma inconjunction with epithelial oncogenesis promotes prostate cancer development Based on our preliminary datawe hypothesize that tumor-derived TNF/TGF1 transcriptionally suppresses AR expression in the stromacausing loss of FGF10 and Wnt16 secretion which are required to maintain the stratified epithelium throughinduction of luminal cells and maintenance of basal cells respectively. To test this we developed the first humanProstate-on-Chip model by culturing basal epithelial cells next to prostate stromal cells within a microfluidicdevice. Within this model we can fully recapitulate the stromal AR-dependent induction of luminal epithelial celldifferentiation. Furthermore co-culturing normal stroma with tumor cells within this model leads to the inductionof CAF phenotypes and reduced stromal AR expression mimicking the tumor/host interactions seen in vivo.Models that recapitulate human glandular organization and its dysregulation during disease development arecritical for our mechanistic understanding of how stroma and oncogenic epithelial interactions drive tumordevelopment. We will test our hypothesis in three aims: 1) Determine the mechanism by which stromal ARmaintains prostate epithelial cell differentiation. Our working hypothesis is that stromal AR signaling inducessecretion of stromal FGF10 and Wnt16 which are required for induction of luminal epithelial cells andmaintenance of basal epithelial cells respectively. 2) Determine the mechanism by which AR expression is lostin the tumor stroma. Our working hypothesis is that tumor-secreted factors TNF and TGF1 acting throughNF-B signaling suppress transcription of the stromal AR gene independent of CAF conversion. 3) Determinethe functional consequence of tumor-induced stromal AR loss on prostate epithelial differentiation in a new denovo in situ human prostate cancer model. Our working hypothesis is that tumor-induced stromal loss of AR-dependent induction of Wnt16 and FGF10 via TNF/TGF1 co-operates with epithelial oncogenes to acceleratetumor development and induce loss of basal epithelial cells. The proposed studies will be the first to demonstratehow TNF/TGF-mediated suppression of stromal AR expression leads to the loss of Wnt16 and FGF10 topromote prostate cancer development. These studies will also provide the framework for further development ofthe first human Prostate-on-Chip model which recapitulates human prostate biology for basic and translationcancer research.