Research article
Breast cancer proteomics reveals correlation between estrogen receptor status and differential phosphorylation of PGRMC1
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* Corresponding author: Michael A Cahill mike.cahill@arcor.de
1 Department of Obstetrics and Gynecology, University of Tuebingen, Calwerstraße, 72076 Tübingen, Germany
2 Current Address: Department of Surgery, Indiana University School of Medicine, W Walnut Street, Indianapolis, Indiana, 46202, USA
3 ProteoSys AG, Carl-Zeiss-Straße, 55129 Mainz, Germany
4 Department of Pathology, University of Tuebingen, Liebermeisterstraße, 72076 Tübingen, Germany
5 Current Address: Department of Pathology, Ludwig-Maximilians-University of Munich, Thalkirchnerstraße, 80337 Munich, Gemany
6 Current Address: Merck-Serono – Global Clinical Development Unit Oncology, Merck KGaA, Frankfurter Straße, 64293 Darmstadt, Germany
7 School of Biomedical Sciences, Charles Sturt University, Wagga Wagga, NSW, 2678, Australia
Breast Cancer Research 2008, 10:R85 doi:10.1186/bcr2155
See related editorial by Craven, http://breast-cancer-research.com/content/10/6/113
Published: 15 October 2008Abstract
Introduction
Breast tumors lacking the estrogen receptor-α (ER-α) have increased incidence of resistance to therapy and poorer clinical prognosis.
Methods
Whole 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.
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 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.
Conclusions
PGRMC1 phosphorylation may be involved in the clinical differences that underpin breast tumors of differing ER status.