Glycan gene expression signatures in normal and malignant breast tissue; possible role in diagnosis and progression

Abstract

Glycosylation is the stepwise procedure of covalent attachment of oligosaccharide chains to proteins or lipids, and alterations in this process have been associated with malignant transformation. Simultaneous analysis of the expression of all glycan-related genes clearly gives the advantage of enabling a comprehensive view of the genetic background of the glycobiological changes in cancer cells. Studies focusing on the expression of the whole glycome have now become possible, which prompted us to review the present knowledge on glycosylation in relation to breast cancer diagnosis and progression, in the light of available expression data from tumors and breast tissue of healthy individuals. We used various data resources to select a set of 419 functionally relevant genes involved in synthesis, degradation and binding of N-linked and O-linked glycans, Lewis antigens, glycosaminoglycans (chondroitin, heparin and keratan sulfate in addition to hyaluronan) and glycosphingolipids. Such glycans are involved in a number of processes relevant to carcinogenesis, including regulation of growth factors/growth factor receptors, cell-cell adhesion and motility as well as immune system modulation. Expression analysis of these glycan-related genes revealed that mRNA levels for many of them differ significantly between normal and malignant breast tissue. An associative analysis of these genes in the context of current knowledge of their function in protein glycosylation and connection(s) to cancer indicated that synthesis, degradation and adhesion mediated by glycans may be altered drastically in mammary carcinomas. Although further analysis is needed to assess how changes in mRNA levels of glycan genes influence a cell's glycome and the precise role that such altered glycan structures play in the pathogenesis of the disease, lessons drawn from this study may help in determining directions for future research in the rapidly-developing field of glycobiology.

Figure 1

Glycosylation in carcinogenesis. Glycosylation plays a principal role in a number of cellular processes of key importance for carcinogenesis. Two metastasizing carcinoma cells are illustrated, entering distant tissue through the blood stream. Six important processes for cancer development and progression (–) influenced by various glycosylation types are indicated; growth receptors (especially EGFR and TβR) are influenced by N‐glycosylation in concert with galectins; growth factors and other signaling molecules may have elevated concentrations, filtered or sequestered by glycosaminoglycans and O‐glycosylated mucins; cell‐cell adhesion might be mediated either directly by for example glycosynapses consisting mainly of glycosphingolipids ‐ or, more importantly, indirectly by modulation of integrins and cadherins by N‐linked glycosylation; O‐glycosylated mucins, both secreted and membrane‐bound, may constitute a physical barrier or act on specific leukocyte receptors thereby modulating immune system response towards the malignant cells; N‐linked glycosylation may enhance motility of transformed cells by regulating integrin functionality; adhesion to endothelium can be mediated by a number of mechanisms, including binding of Lewis antigens by endothelial selectins.

Figure 2

N‐linked glycosylation. (For symbol explanation, see Figure 7) A) Initial steps of N‐glycan synthesis pathway. First, two GlcNAcs are added to the Dol‐PP anchor in the outer leaflet of ER's lipid membrane. Further, five mannose saccharides are attached to the structure. This precursor located in cytosol, is now flipped by a yet not fully elucidated mechanism to the inner leaflet. For the rest of the synthesis process, this glycan structure is situated inside the ER lumen. Here, four more mannoses as well as three glucoses are added to create the mature N‐glycan precursor. Genes encoding transferases that are responsible for these reactions are designated ALG (asparagine‐linked glycosylation). The mature precursor is then detached from its dolichol anchor and transferred to a target polypeptide sequence co‐translationally by the large enzyme complex, OST (modified from Varki et al., 2009). B) N‐glycan branching. After being transferred to a protein, the N‐glycan goes through glucose and mannose trimming, the former being involved in polypeptide folding quality control. The resulting Man₅GlcNAc₂ structure may be branched – a process mediated by the Mgat family of GlcNAc‐transferases. Up to four branches can be added by Mgat1, ‐2, ‐4 and 5 respectively and further elongated (orange arrows). Of these, Mgat5 appears to be the most interesting in carcinogenesis; the branch it initiates is preferentially elongated by polylactosamine. In addition to the four previously mentioned branches a so‐called bisecting β‐3 branch may be added by Mgat3. This bisecting GlcNAc terminates all further branching, including that mediated by Mgat5. Thus, activity of Mgat3 might inhibit polylactosamine synthesis. The two key reactions, performed by Mgat3 and Mgat5, are highlighted in red. The Mgat transferases require UDP‐GlcNAc which is imported through a transporter (SLC35A3). Genes encoding relevant transferases are displayed in black italic font (modified from Lau and Dennis, 2008). C) Core fucosylation. This is one of the possible modifications made to N‐glycans' core structure.

Figure 3

O‐linked glycosylation. (For symbol explanation, see Figure 7) A) Synthesis of O‐linked glycans. Several different core O‐glycan structures exist, but some are very rare. The initial synthesis of cores 1 and 2 as well as tumor‐associated antigens (T antigens) are illustrated. Each of the cores can be extended and further modified by for example fucosylation (not shown). Genes encoding relevant transferases are displayed in black italic font (modified from Varki et al., 2009; Tarp and Clausen, 2008). B) Initiation of O‐glycosylation on MUC1. This illustration shows a single VNTR (orange) of MUC1 with its amino acid sequence, and specificities of the well‐studied GalNAcTs. The enzymes involved are sometimes overlapping in specificity, although it should be noted that some transferases require preceding activity of other GalNAcTs (not shown). It is also worth mentioning that other transferases from this family are known to act on MUC1 although their specificity is yet to be defined (Bennett et al., 1999a,b) (modified from Tarp and Clausen, 2008).

Figure 4

Lewis antigens. (For symbol explanation, see Figure 7) Lewis antigens are usually subdivided into two groups – type 1 and 2 – depending on whether the terminal galactose is bound to the preceding GlcNAc by a β3 or β4 bond (in red font). Epitopes in the latter category are considered as tumor‐associated antigens. Both type 1 and 2 structures may appear on a variety of glycans (denoted as “R”). Therefore they are important in interaction with other cells like endothelial cells. The main Lewis antigens are shown, alongside genes encoding key transferases in their synthesis (modified from Varki et al., 2009).

Figure 5

Ganglio‐series glycosphingolipid synthesis pathway. (For symbol explanation, see Figure 7) Key genes involved in the pathway are indicated, and four have been denoted numerically to indicate that the sialyltransferases encoded by these genes catalyze several steps of the pathway (modified from Varki et al., 2009).

Figure 6

Structure of glycosaminoglycans (GAGs). (For symbol explanation, see Figure 7) The three main structural aspects of GAGs are illustrated; attachment to core proteins, the repeating disaccharide sequence, and the hallmark sulfation. Attachment to protein cores is mediated by different glycan structures, except for hyaluronan which is not covalently bound to any other molecule. Chondroitin and heparan sulfate share a common tetrasaccharide core structure while keratan sulfate may be attached to either N‐linked or O‐linked glycans (left). Further, GAGs consist of repeated disaccharide units which are unique for each glycosaminoglycan type (middle). Addition of sulfate is typical for all GAGs except hyaluronan. Possible sulfation patterns for one of the glycosaminoglycans, chondroitin sulfate, are shown to the right (modified from Varki et al., 2009; Sugahara et al., 2003).

Figure 7

Symbols. The different symbols used to denote the various sugar moieties in 2, 3, 4, 5, 6 are shown. The orange rectangle illustrates a portion of a polypeptide with the mid part representing the amino acid to which glycan structures are attached.

Figure 8

Summary of sources used to compile the glycan gene list. Bold arrow indicates the main source.

Figure 9

Hierarchical clustering of glycan gene expression. A) Data set A. Malignant tissue and samples from healthy women are marked red and green on the dendrogram, respectively. These two groups are well separated indicating that glycan gene expression is profoundly altered during carcinogenesis. A subgroup of normal samples appears to have a different glycan gene signature. This group is largely identical to the “cluster 1” group defined by whole‐genome expression analysis by Haakensen et al. (unpublished data). Samples classified as “cluster 1” are marked blue. B) Data set B. Tumor tissue samples are marked red on the dendrogram – bright red if extracted from a tumor biopsy or dark red in case whole‐tumor was used. Core biopsies of adjacent breast tissue are marked green. The malignant and the non‐malignant samples are well separated in this data set as well. Note that the normal and malignant samples are equally well separated in both data sets (with only a few misplaced samples) despite that data set A contains samples from different individuals while data set B consists of tumor and adjacent normal tissue from the same patient. These heatmaps with sample names and gene symbols can be found in supplementary material Figure 1A and B.