Discovery of prognostic biomarkers from the prostate cancer membrane proteome using bioorthogonal chemistry.


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Lynn Asante-Asare1,Mike Deery2,Renata Feret2,Yagnesh Umrania2,Jodi Miller1,Cara Brodie1,Jo Arnold1,André Neves1,Kevin Brindle1
1Cancer Research UK Cambridge Institute,2Cambridge Centre for Proteomics

Abstract

Background

The link between aberrant glycosylation and cancer progression has formed the basis for using glycoproteins and specific polysaccharide structures in the clinical management of cancer. However, the clinical utility of serum biomarkers, such as PSA, have been challenged in recent years due to low cancer specificity and high false-positive rates. The aim of this study was to use synthetic analogues of monosaccharides called azidosugars, to dynamically monitor changes in cell surface glycosylation in a model of prostate cancer progression, in order to identify novel biomarkers.

Method

A panel of human prostate cancer cell lines was used, which represented the stages of prostate cancer progression, in vitro. The cell lines were incubated with synthetic analogues of sialic acid and N-acetylgalactosamine that had been labelled with an azide group (N-azidoacetylmannosamine (Ac4ManNAz) and N-azidoacetylgalactosamine (Ac4GalNAz)). These are incorporated biosynthetically into cell surface glycans. The membrane proteome was isolated by detergent-based fractionation, the incorporated azide groups covalently ligated to Alkyne-biotin using copper-catalysed chemistry, and the azidosugar labelled proteins enriched using streptavidin-coupled beads. Proteins were resolved via gel electrophoresis and identified by mass spectrometry.

Results

Proteomic analysis revealed 8 proteins exhibiting an increase in Ac4ManNAz and Ac4GalNAz glycan labelling, which correlated with an increase in the metastatic potential of the cell lines. Normalised Spectral Abundance Factors, showed, as expected, a relative increase in the expression of total PSA, but also an increase in Ac4ManNAz and Ac4GalNAz glycan labelling, which correlated with an increase in the metastatic potential.  More interesting was the case of Basigin (CD147), which showed a strong increase in Ac4ManNAz and Ac4GalNAz labelling, despite its total expression decreasing across the panel of cell lines.

Conclusion

Our data suggest that differential sialyation and N-acetylgalactosamine glycosylation of proteins may be a more sensitive and specific marker for monitoring prostate cancer progression, than increases in total protein expression alone.