Estrogen receptor-a positive (ERα+) breast cancer (BC) represents 70-80% of newly diagnosed cases. While potentially responsive to anti-estrogens, progression of ERα+ BC to anti-estrogen refractory disease in the metastatic setting is a common occurrence. Endocrine resistance mechanisms may differ between subtypes of ERα+ BC and involve both ERα-independent and ERα-dependent forms that remain poorly understood. Prolactin (PRL) interacts with estrogens to stimulate growth of certain subtypes of ERα+ BC. At the same time, the PRL-Jak-Stat5 pathway promotes differentiation and inhibits invasive features of BC, and loss of Stat5a signaling in a subgroup of ERα+ BC is associated with anti-estrogen therapy failure. The involvement of PRL pathways in growth and progression of anti-estrogen refractory BC subtypes remains to be determined. There is a lack of preclinical human ERα+ BC models that recapitulate progression from localized mammary gland growth to distant metastasis. Xenografts of patient-derived ERα+ BC exhibit poor take rate in mice. We have discovered that murine PRL is a poor agonist and a potent antagonist for human PRL receptor (PRLr). Bovine PRL is also a poor agonist with antagonist activity for human PRLr. Laboratory human BC lines therefore have been selected for PRL-independent growth and may only represent subtype(s) of ERα+ BC. To address this problem, we generated hPRL knock-in mice in the immunodeficient Nod-Scid-IL2Rγ strain that express physiological levels of circulating hPRL. Remarkably, PRL-humanized mice display greatly increased take rate of patient-derived xenografts of ERα+ BC. We established a novel panel of serially transplantable ERα+ Luminal B BC lines, several of which spontaneously metastasize to lungs and liver. The distant metastases become anti-estrogen refractory despite continued expression of ERα+, but show PRL-dependence. Our long-range goal is to determine mechanisms of anti-estrogen refractoriness of BC to improve clinical management. Aim 1 is focused on a recently identified Luminobasal subtype of ERα+ BC (ERα+/CK5+). Aim 1 explores a distinct ERα-independent mechanism of anti-estrogen refractory BC in pre-existing ERα+ laboratory cell lines. We hypothesize that in Luminobasal BC, loss of PRL-Stat5 signaling promotes loss of ERα and subsequent anti-estrogen resistance due to defective Stat5a-driven differentiation. Aim 2 is focused on the Luminal B BC subtype (ERα+/CK5-/Ki67high) and is centered on our new PRL-dependent patient-derived xenograft lines. Aim 2 will test the hypothesis that in Luminal B BC, unlike in Luminobasal BC, PRL facilitates growth and survival of metastases and that PRLr-pathway targeting cooperates with anti-estrogens to eliminate distant metastases.