Integration of genetic and metabolic features related to sialic acid metabolism distinguishes human breast cell subtypes

Abstract

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.

Cell Line-, Protein-, and Sialoglycosite-Specific Control of Flux-Based Sialylation in Human Breast Cells: Implications for Cancer Progression

“…Once synthesized and dephosphorylated, sialic acid enters the nucleus where CMAS converts it to the corresponding nucleotide sugar (e.g., CMP-Neu5Ac); CMP-Neu5Ac is transported into the Golgi apparatus by SLC35A1 and SLC35A3 where a subset (depending on the cell line) of the 20 human STs create sialoglycoconjugates (primarily, N-and O-linked glycoproteins or gangliosides [i.e., sialic acid-modified glycosphingolipids]). (B) Representative results from a previous study (Saeui et al, 2018) are shown to illustrate the minimal (in the MCF-10A line) to large increase (in the T-47D line) that occurs in flux-driven sialylation in human breast cell lines (note that statistical significance for similar results is provided in Figure 2). (C) The focus of the current paper is to conduct a detailed "glycosite" analysis of cell surface glycoproteins obtained from 1,3,4-O-Bu 3 ManNAc treated cells.…”

“…Each experiment was performed in triplicate and cell counts were normalized to untreated controls. Intracellular free and conjugate bound sialic acid levels were determined for treated (100 µM) and untreated cells using the periodate resorcinol method as previously described (Jourdian et al, 1971;Yarema et al, 2001;Saeui et al, 2018).…”

“…Although limited to a single cell line, these results unambiguously showed that metabolic flux can influence sialylation; cell surface sialic acid increased globally by ∼75% and individual glycosites increased by as much as ∼8-fold. Subsequent studies expanded our analyses to multiple cell lines and to a different type of cancer by comparing intracellular sialic acid production in different subtypes of breast cancer (Saeui et al, 2018). Certain results FIGURE 1 | Overview of ManNAc analog-based control of flux through the sialic acid biosynthetic machinery.…”

“…(C) The focus of the current paper is to conduct a detailed "glycosite" analysis of cell surface glycoproteins obtained from 1,3,4-O-Bu 3 ManNAc treated cells. This figure is adapted from our previous publication (Saeui et al, 2018).…”

Advanced Bottom‐Up Engineering of Living Architectures

“…Because these probes are not transferable across cells, the addition of different reporters at various time intervals allow for differential labeling of tissue layers and display time‐sensitive imaging of GTCs development. Another unique feature of glycoengineering is the sensitivity to different intracellular metabolisms that can distinguish cell subtypes and thus monitor stages of human breast cancer . Similarly, incorporation of azido‐sugars was significantly increased during cardiac hypertrophy .…”