Identifying N-Glycan Biomarkers in Colorectal Cancer by Mass Spectrometry

Abstract

Colorectal cancer (CRC) is one of the most prevalent cancers worldwide. Delineating biological markers (biomarkers) for early detection, when treatment is most effective, is key to prevention and long-term survival of patients. Development of reliable biomarkers requires an increased understanding of the CRC biology and the underlying molecular and cellular mechanisms of the disease. With recent advances in new technologies and approaches, tremendous efforts have been put in proteomics and genomics fields to deliver detailed analysis of the two major biomolecules, genes and proteins, to gain a more complete understanding of cellular systems at both genomic and proteomic levels, allowing a mechanistic understanding of the human diseases, including cancer, and opening avenues for identification of novel gene and protein based prognostic and therapeutic markers. Although the importance of glycosylation in modulating protein function has long been appreciated, glycan analysis has been complicated by the diversity of the glycan structures and the large number of potential glycosylation combinations. Driven by recent technological advances, LC-MS/MS based glycomics is gaining momentum in cancer research and holds considerable potential to deliver new glycan-based markers. In our laboratory, we investigated alterations in N-glycosylation associated with CRC malignancy in a panel of CRC cell lines and CRC patient tissues. In an initial study, LC-MS/MS-based N-glycomics were utilized to map the N-glycome landscape associated with a panel of CRC cell lines (LIM1215, LIM1899, and LIM2405). These studies were subsequently extended to paired tumor and nontumorigenic CRC tissues to validate the findings in the cell line. Our studies in both CRC cell lines and tissues identified a strong representation of high mannose and α2,6-linked sialylated complex N-glycans, which corroborate findings from previous studies in CRC and other cancers. In addition, certain unique glycan determinants such as bisecting β1,4-GlcNAcylation and α2,3-sialylation, identified in the metastatic (LIM1215) and aggressive (LIM2405) CRC cell lines, respectively, were shown to be associated with epidermal growth factor receptor (EGFR) expression status. In this Account, we will describe the mass spectrometry based N-glycomics approach utilized in our laboratory to accurately profile the cell- and tissue-specific N-glycomes associated with CRC. We will highlight altered N-glycosylation observed by our studies, consistent with findings from other cancer studies, and discuss how the observed alterations can provide insights into CRC pathogenesis, opening new avenues to identify novel disease-associated glycan markers.

Diagnostic Potential of Transferrin Glycoforms—A Lectin‐Based Protein Microarray Approach

“…To study the influence of aging, two old‐aged groups were formed, up to and above 80 years old, and in each group two subgroups were defined: male and female. Two groups of patients and their hTf were included in the study as well, as it was reported that both T2DM and CRC may alter protein glycosylation . The results for interactions between hTf molecules of different origin and six most reactive lectins are shown in Figure and statistical analysis of the data (at the level of technical replicates needed for obtaining the information on the reproducibility of the created analytical platform …”

“…This finding is also in accordance with the results of Qiu et al [9] who reported an increase in core fucosylation of several serum proteins due to colon cancer, such as complement C3, histidine-rich glycoprotein, and kininogen-1. [11,38,51,52]…”

“…Besides hTf concentration, the pattern of hTf glycosylation may be altered due to a disease or a condition, such as congenital disorder of glycosylation (CDG), alcohol abuse, Alzheimer's or Parkinson's disease, arthritis, anemia, and cancer . Investigation of hTf glycosylation pattern can aid in the diagnosis of genetic or metabolic disturbances, help in the follow‐up of therapeutic treatments and it can assist in investigation of physiological interactions between hTf and human lectins in body fluids or on cells (e.g., receptors) …”

Tumor-suppressive miR-26a and miR-26b inhibit cell aggressiveness by regulating FUT4 in colorectal cancer

“…Several CRC tissue-associated changes in glycans have been reported and recently reviewed. 15 Holst et al 16 showed pronounced differences between the N -glycosylation patterns of CRC cell lines, and CRC cell line profiles differed from tissue-derived N -glycan profiles. Osuga et al 17 found that increased fucosylation was correlated with metastatic potential in CRC.…”

Recent Advances in the Mass Spectrometry Methods for Glycomics and Cancer

“…PGC stationary phase in LC-MS was used by Sethi et al to determine the reduced N-glycans on cell membrane from cell lines of colorectal cancer. 64 In the study, increase of high mannose glycans and α(2,6)-sialylation was observed for the cancer. Song et al has used this method to determine the relative abundances of N-glycans released from serum (Figure 6) as well as biological variations between individuals.…”