Abstract
IntroductionBreast tumors lacking the estrogen receptor-α (ER-α) have increased incidence of resistance to therapy and poorer clinical prognosis.MethodsWhole tissue sections from 16 cryopreserved breast cancer tumors that were either positive or negative for the ER (eight ER positive and eight ER negative) were differentially analyzed by multiplex imaging of two-dimensional PAGE 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.ResultsProteins 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 ER-negative basal epithelial cells of mammary ductules. Even in advanced tumors, high levels of ER or PGRMC1 were almost mutually exclusive in individual cells. In five out of five examined ductal in situ breast cancers of comedo type, PGRMC1 was expressed in glucose transporter 1 negative or positive poorly oxygenated cells surrounding the necrotic core, surrounded by a more distal halo of ER-positive cells.ConclusionsPGRMC1 phosphorylation may be involved in the clinical differences that underpin breast tumors of differing ER status.
Highlights
Breast tumors lacking the estrogen receptor-α (ER-α) have increased incidence of resistance to therapy and poorer clinical prognosis
The classical estrogen receptor (ER)-α is found in 50% to 80% of breast tumors and ER-α status is essential in making clinical decisions about endocrine therapy with anti-estrogens, which inhibit the mitogenic activity of estrogens in breast cancer
There were four differential comparisons, each performed in inverse replicate 54 cm serial isoelectric focusing (IEF)-immobilized pH gradient (IPG) to generate eight IEF samples and 24× two-dimensional PAGE gels, which were differentially quantified by ProteoTope imaging
Summary
Breast tumors lacking the estrogen receptor-α (ER-α) have increased incidence of resistance to therapy and poorer clinical prognosis. The classical estrogen receptor (ER)-α is found in 50% to 80% of breast tumors and ER-α status is essential in making clinical decisions about endocrine therapy with anti-estrogens, which inhibit the mitogenic activity of estrogens in breast cancer. When ER-α-positive cells are implanted in nude mice, tumors appear only in the presence of estrogens and are poorly metastatic as compared with those developed from ER-α-negative breast cancer cell lines [9,10]. This paradox suggests that ER-α expression could be associated with or involved in pathways that hinder cancer progression
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