Glycomic Approaches for the Discovery of Targets in Gastrointestinal Cancer

Abstract

Gastrointestinal (GI) cancer is the most common group of malignancies and many of its types are among the most deadly. Various glycoconjugates have been used in clinical practice as serum biomarker for several GI tumors, however, with limited diagnose application. Despite the good accessibility by endoscopy of many GI organs, the lack of reliable serum biomarkers often leads to late diagnosis of malignancy and consequently low 5-year survival rates. Recent advances in analytical techniques have provided novel glycoproteomic and glycomic data and generated functional information and putative biomarker targets in oncology. Glycosylation alterations have been demonstrated in a series of glycoconjugates (glycoproteins, proteoglycans, and glycosphingolipids) that are involved in cancer cell adhesion, signaling, invasion, and metastasis formation. In this review, we present an overview on the major glycosylation alterations in GI cancer and the current serological biomarkers used in the clinical oncology setting. We further describe recent glycomic studies in GI cancer, namely gastric, colorectal, and pancreatic cancer. Moreover, we discuss the role of glycosylation as a modulator of the function of several key players in cancer cell biology. Finally, we address several state-of-the-art techniques currently applied in this field, such as glycomic and glycoproteomic analyses, the application of glycoengineered cell line models, microarray and proximity ligation assay, and imaging mass spectrometry, and provide an outlook to future perspectives and clinical applications.

Keywords: colorectal cancer; gastric cancer; glycan biomarkers; glycomics; imaging mass spectrometry; microarray; pancreatic cancer; proximity ligation assay.

Figure 1

Schematic depiction of glycan alterations during malignant transformation. Healthy epithelium displaying cell polarity, organized glycoprotein localization, and normal glycosylation pattern. Malignant cells with misslocalized glycoproteins and altered expression of genes involved in glycosylation pathways leading to the aberrant expression of glycan moieties. The glycosyltransferases involved in glycan alterations during malignant transformation are listed.

Figure 2

Glycomic and glycoproteomic strategies for glycan analysis. (A) Glycomic analysis. N- and O-glycans are PNGase F or chemically released, respectively. Once the glycans are released, they are structurally analyzed (usually by mass spectrometry). (B) Glycoproteomic analysis. The glycoproteins are either purified from a complex mixture and then enzymatically digested (1) or first digested and then purified (2). Method 1 will result in glycopeptides and non-glycosylated peptides originated solely from glycoproteins. Method 2 will result in glycopeptides only. These glycopeptides can be directly analyzed to generate site-specific glycan structures. Alternatively, the glycans of the glycopeptides can be released as described in (A) and the peptides are analyzed.

Figure 3

Arrays for glycan analysis. (A) Glycan arrays. Different glycan moieties are spotted onto the array to determine specificities of labeled glycan-binding molecules (typically lectins or antibodies). (B) Glycoprotein arrays. Glycoproteins are enriched from a sample and spotted onto the array. The glycosylation moieties of the spotted glycoproteins are determined by screening with different glycan-binding molecules. (C) Lectin arrays. A series of lectins with well-defined glycan-binding properties are spotted onto the array and different labeled proteins are tested. (D) Antibody-lectin sandwich arrays. Multiple antibodies for a series of proteins of interest are spotted onto the array, and the glycan epitopes of the captured proteins are probed using labeled lectins and glycan-binding antibodies.

Figure 4

In situ proximity ligation assay. (A) Two antibodies, one specific for the protein backbone and another specific for a glycan epitope, are conjugated with two different oligonucleotide chains (PLA probes). (B) In case the antibodies bind to molecules in close proximity a bridging sequence links the two oligonucleotide sequences. (C) A subsequent polymerase induced amplification in combination with labeled nucleotides leads to the formation of a fluorescent or chromogenic signal at the co-expression site of the protein and glycan, allowing the in situ detection of the PLA signal.

Figure 5

Imaging mass spectrometry. MALDI-based ionization generates an MS glycan spectrum of a selected spot of a tissue section. This process is repeated throughout the tissue. Identified glycan structures are mapped on the tissue and can be correlated with histopathological features.