Increased mammographic density is associated with increased incidence of breast cancer, and is largely attributed to increased levels of stromal collagen and changes in the composition of the extracellular matrix.  These changes result in a stroma that is mechanically stiffened, triggering signaling pathways that lead to tumor development, invasion and metastasis.  In addition, we find that aligned collagen fibers facilitate invasion, correlate with metastasis, and serve as a prognostic signature for poor outcome in patients.  While hormones, including prolactin and estrogen, have been associated with elevated mammographic density in patients and are independently associated with the development of estrogen receptor positive (ER?+) breast cancer, little is known about the interplay of hormones and extracellular matrix composition and organization in the development, progression and therapeutic resistance of breast cancer.  Moreover, although prolactin exposure is associated with higher risk of metastases and poor long term survival of ER?+ cancers, the best studied prolactin-activated mediator, STAT5, is associated with anti-estrogen sensitivity and better differentiated tumors.  Our preliminary data demonstrate that elevated collagen density alone can shift the spectrum of PRL-induced signals away from STAT5 and toward ERK1/2, which is associated with pro-proliferation/invasion signals, and reduces estrogen responsiveness.  Based on these findings, in this revised proposal, we will address the hypothesis that changes in the density, composition, and alignment of the ECM switch the balance of hormonal signals from pro-differentiation to pro-tumorigenic, which further alters the stroma in a feed-forward manner to promote breast cancer progression.  In Aim 1, we will utilize a defined 3D culture system, mechanically precise collagen-coated polyacrylamide substrata, and aligned collagen matrices to examine effects in hormonally responsive breast carcinoma cells on prolactin and estrogen-induced signals and behavioral outcomes.  In Aim 2, we will examine the interplay between carcinoma-associated fibroblasts, normal mammary fibroblasts, hormones and tumor cells in vitro.  In Aim 3, we will extend these studies in vivo, examining these interactions on disease progression, metastasis and responsiveness to anti-estrogen therapy in xenograft models, immunocompetent mouse models and clinical tumors.  Although many ER?+ cancers are successfully treated by anti-estrogen therapies, nearly 25% display initial or acquired resistance and thereby constitute the majority of breast cancer mortality.  Understanding the interactions of the extracellular environment on hormonal signals will illuminate the contribution of these factors to breast pathology and therapeutic responsiveness, and point to novel treatment opportunities.