In this report we use 'high-flux' tributanoyl-modified
N-acetylmannosamine (ManNAc) analogs with natural N-acetyl as well as non-natural azido- and
alkyne N-acyl groups (specifically, 1,3,4-O-Bu3ManNAc, 1,3,4-O-Bu3ManNAz, and 1,3,4-O-Bu3ManNAl respectively) to probe intracellular
sialic acid metabolism in the near-normal MCF10A human breast cell line in comparison with earlier stage T-47D and more advanced stage MDA-MB-231
breast cancer lines. An integrated view of
sialic acid metabolism was gained by measuring intracellular
sialic acid production in tandem with transcriptional profiling of genes linked to
sialic acid metabolism. The transcriptional profiling showed several differences between the three lines in the absence of ManNAc analog supplementation that helps explain the different sialoglycan profiles naturally associated with
cancer. Only minor changes in
mRNA transcript levels occurred upon exposure to the compounds confirming that metabolic flux alone can be a key determinant of sialoglycoconjugate display in
breast cancer cells; this result complements the well-established role of genetic control (e.g., the transcription of STs) of sialylation abnormalities ubiquitously associated with
cancer. A notable result was that the different cell lines produced significantly different levels of
sialic acid upon exogenous ManNAc supplementation, indicating that feedback inhibition of
UDP-
GlcNAc 2-
epimerase/ManNAc
kinase (GNE)-generally regarded as the 'gatekeeper'
enzyme for titering flux into
sialic acid biosynthesis-is not the only regulatory mechanism that limits production of this
sugar. A notable aspect of our metabolic glycoengineering approach is its ability to discriminate cell subtype based on intracellular metabolism by illuminating otherwise hidden cell type-specific features. We believe that this strategy combined with multi-dimensional analysis of
sialic acid metabolism will ultimately provide novel insights into
breast cancer subtypes and provide a foundation for new methods of diagnosis.