Increased wound-response revealed by proteomics of estrogen receptor-negative breast cancers Free

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Background:

The classical estrogen receptor α (ER) is found in 50-80% of breast tumors. While the mitogenic action of estrogens in breast cancer cells is well established, there is also evidence that estrogen receptors mediate protective, anti-invasive effects. Therefore, downstream ER-dependent proteins should be identified as possible targets to investigate and modify the mechanism of this phenomenon.

Aim:

The aim of this project was to identify proteins differentially expressed in fresh-frozen ER-positive and -negative breast cancer.

Methods:

Whole tissue sections from eight ER-positive and eight ER-negative cryo-preserved breast cancer specimen were lysed and sub-pooled according to ER status into pools of two with normalised equal amounts of protein. These were analysed by differential multiplex ProteoTope imaging of 2D-PAGE gels using 54 cm isoelectric focussing. This method is based on labeling proteins with chemically identical iodine radio-isotopes (I-125, I-131) and provides a direct quantitative and multi color differential proteome display at the low-attomole level. Spots were matched across gels, and their intensities were analysed relative to ER status. The statistically most significant differential protein spots were identified by mass spectrometry using picked co-migrating non-radioactive tumor proteins.

Results:

In total, proteins from 325 spots were identified by MALDI-TOF PMF with MASCOT scores greater than 70. 72 spots represented 16 proteins that were identified in more than one protein spot. Proteins significantly differentially abundant across all pools at the 0.1% level were consistent with previously published literature. We observed elevated levels of keratins 19, 18 and 8 in ER-positive tumors suggesting a luminal phenotype for ER-positive tumors. Additionally, proteins associated with increased inflammation (immunoglobulin kappa light chain) and wound response (vimentin, apolipoprotein A1, cyclophin A, transferrin, carbonic anhydrase, and PGRMC1) were observed in ER-negative tumors. The most highly differential abundant protein in ER-negative tumors relative to ER-positive tumors in our study was fibrinogen gamma A chain, followed by fibrin. Cancer-related fibrin deposition and fibrinolysis characterise many solid tumours, with cancer cells supplying many of the functions supplied by platelets in normal blood clotting. The deposition of fibrinogen without subsequent conversion to fibrin in the tumour stroma is reportedly a hallmark of breast carcinoma.

Conclusion:

We demonstrate the applicability of ProteoTope, high resolution 2D-PAGE and sample pooling strategies for the discovery-directed differential quantification of complex proteomes from clinical samples. Our data are consistent with previously published literature, suggesting an altered keratin pool associated with increased inflammation and wound response in ER-negative tumors.