ABSTRACT: INTRODUCTION: The prolactin-Janus-kinase-2-signal transducer and activator of transcription-5 (JAK2-STAT5) pathway is essential for the development and the functional differentiation of the mammary gland. The pathway also has important roles in mammary tumourigenesis. Prolactin regulated target genes are not yet well defined in tumour cells, and we undertook the first large genetic screen of breast cancer cells treated with or without exogenous prolactin. We hypothesize that the identification of these genes should yield insights into the mechanisms by which prolactin participates in cancer formation or progression and possibly how it also regulates normal mammary gland development. METHODS: We used subtractive hybridization to identify a number of prolactin-regulated genes in the human mammary carcinoma cell line SKBR3. Northern analysis and luciferase assays identified the gene encoding heat shock protein 90-alpha (HSP90A) as a prolactin-JAK2-STAT5 target gene, whose function was characterized using apoptosis assays. RESULTS: We identified a number of new prolactin-regulated genes in breast cancer cells. Focusing on HSP90A, we determined that prolactin increased HSP90A mRNA in cancerous human breast SKBR3 cells and that STAT5B preferentially activated the HSP90A promoter in reporter gene assays. Both prolactin and its downstream protein effector, HSP90-alpha, promote survival, as shown by apoptosis assays and by the addition of the heat shock protein 90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), in both untransformed HC11 mammary epithelial cells and SKBR3 breast cancer cells. The constitutive expression of HSP90A, however, sensitized differentiated HC11 cells to starvation-induced wild-type p53-independent apoptosis. Interestingly, in SKBR3 breast cancer cells, HSP90-alpha promoted survival in the presence of serum but appeared to have little effect during starvation. CONCLUSIONS: In addition to identifying new prolactin regulated genes in breast cancer cells, we found that prolactin-JAK2-STAT5 induces expression of the HSP90A gene, which encodes the master chaperone of cancer. This identifies one mechanism by which prolactin contributes to breast cancer. Increased HSP90-alpha expression in breast cancer is correlated with increased cell survival and poor prognosis and HSP90-alpha inhibitors are being tested in clinical trials as a breast cancer treatment. Our results also indicate that HSP90-alpha promotes survival depending upon the cellular conditions and state of cellular transformation.