INTRODUCTION: Breast tumors lacking the estrogen receptor alpha have increased incidence of resistance to therapy and poorer clinical prognosis.METHODS: Whole tissue sections from sixteen cryopreserved breast cancer tumors that were either positive or negative for the estrogen receptor (8x estrogen receptor positive and 8x estrogen receptor negative) were differentially analyzed by multiplex ProteoTope imaging of two dimensional polyacylamide gel electrophoresis gels using 54 cm isoelectric focusing. Differentially detected spots of Progesterone Receptor Membrane Component 1 (PGRMC1) were shown to differ in phosphorylation status by differential two dimensional polyacrylamide gel electrophoresis of phosphatase-treated tumor proteins. Site directed mutagenesis was used to create putative phosphorylation site point mutants in PGRMC1. Stable transfectants of these mutants in MCF7 cells were assayed for their survival after oxidative stress, and for AKT kinase phosphorylation. Immune fluorescence using anti-PGRMC1 monoclonal antibody 5G7 was performed on breast cancer tissue microarrays.RESULTS: Proteins significantly differentially abundant between estrogen receptor negative and estrogen receptor positive tumors at the 0.1% level were consistent with published profiles, suggesting an altered keratin pool, and increased inflammation and wound responses in estrogen receptor negative tumors. Two of three spots of PGRMC1 were more abundant in estrogen receptor negative tumors. Phosphatase treatment of breast tumor proteins indicated that the PGRMC1 isoforms differed in their phosphorylation status. Simultaneous mutation of PGRMC1 Serine 56 and Serine 181 fully abrogated the sensitivity of stably transfected MCF7 breast cancer cells to peroxide-induced cell death. Immune fluorescence revealed that PGRMC1 was primarily expressed in estrogen receptor negative basal epithelial cells of mammary ductules. Even in advanced tumors high levels of estrogen receptor or PGwere almost mutually exclusive in individual cells. In 5/5 examined ductal in situ breast cancers of comedo-type, PGRMC1 was expressed in Glucose Transporter 1 (GLUT-1)-negative or GLUT-1-positive poorly oxygenated cells surrounding the necrotic core, surrounded by a more distal halo of estrogen receptor positive cells.CONCLUSIONS: PGRMC1 phosphorylation may be involved in the clinical differences underpinning breast tumors of differing estrogen receptor status.