Glycosylation in Cancer

Prostate cancer

Worldwide, more than a million new prostate cancer (PCa) cases were diagnosed in 2012, which means, that after lung cancer, PCa is the most prevalent cancer in men. Currently, the protein concentration of prostate specific antigen (PSA) that is circulating in the blood stream is used as an early screening method of PCa. This test was approved in 1994 by the FDA, where elevated levels of the PSA (> 3 ng/mL) could indicate that the patient has PCa. However, studies revealed that the test lacks specificity as the PSA concentration is not only dependent on PCa but could also be elevated through other cases, resulting in unnecessary biopsies. For example, inflammation of the prostate (prostatitis), an enlarged prostate (benign hyperplasia, BPH) or even cycling can result in higher PSA concentrations in the serum. More importantly, PSA is not able to distinguish between aggressive and non-aggressive tumours. Hence, there is an urgent need for more specific biomarkers.

Literature reveals that the glycosylation of PSA could play an important role in the development of a more specific test. It is known that PSA contains a single, very heterogeneous N-linked glycosylation site which has been studied in the past. Nonetheless, specific details regarding molecular features (e.g. core fucosylation or the complexity of the antennae) and their potential for improving the PCa diagnosis has not yet been studied.

Our research group studies the glycosylation of PSA1,2 and its relevance for a more specific biomarker in collaboration with the Urology department of the Amsterdam Medical Center (AMC), under supervision of Prof. Dr. Manfred Wuhrer (LUMC), Prof. Dr. Jean J.M.C.H. de la Rosette (AMC) and Dr. Theo M. de Reijke (AMC). The analytical platforms that are used for this analysis are CE-ESI-MS and MALDI-TOF/MS. This project is funded by Astellas.

Conventional analytical approach for the analysis of a patients’ sample.

 

Colorectal cancer

One of the most important modification of proteins and lipids is glycosylation. Glycan alterations have been shown to be involved in the malignant transformation and progression in many tumors, predominantly in Colorectal Cancer (CRC).3 Our group made an important contribution in understanding the (glyco-)biology of CRC through profiling the differential N-glycan and glycosphingolipid (GSL)glycan signatures of colorectal cancer vs. paired control tissues by mass spectrometry techniques.4,5 In particular, the combination of Matrix-Assisted Laser Desorption Time-of-Flight Mass Spectrometry (MALDI-TOF-MS) with Hydrophilic Interaction Liquid Chromatography (HILIC) or 2-dimensional Liquid Chromatography (2D-LC-MS/MS) has been shown to be a promising technique for the identification of N-/GSLglycosylation alterations that might contribute to the discovery of new biomarkers.4,5 These findings, together with the recent N-glycosylation profiling of 25 different CRC cell lines,6 set the basis for our current study on CRC associated glycan changes. These are further explored as diagnostic or prognostic markers, as well as as novel therapeutic target in this tumor. On-going collaborations with the Department of Surgery and the Department of Pathology of the LUMC, but also external partners will advance our knowledge of the (glyco-)biology of this cancer, with the ultimate goal of developing novel clinical assays. Recent advances in MALDI-mass spectrometry imaging (MSI) by our group together with the Imaging group of the LUMC and external partners add a valuable new tool for the spatial characterization of N-glycans and proteins in tissue.7,8 Furthermore, as coordinator and partner of the Marie Curie European Training Network GlycoCan, we are exploring differentially expressed and/or glycosylated surface and secreted proteins in a set of CRC cell lines as well as the aberrant glycosylation of different glycoproteins such as CEA.

  1. Kammeijer G.S.M. et al. Dopant Enriched Nitrogen Gas Combined with Sheathless Capillary Electrophoresis–Electrospray Ionization-Mass Spectrometry for Improved Sensitivity and Repeatability in Glycopeptide Analysis. Anal. Chem. 88(11), 5849-56 (2016).
  2. Kammeijer G.S.M. et al. Sialic acid linkage Analysis of Glycopeptides using CE-ESI-MS(/MS). Sci. Rep. 7, 3733 (2017).
  3. Holst, S., Wuhrer, M. & Rombouts Y. Chapter Six-Glycosylation Characteristics of Colorectal Cancer. Advances Cancer Res. 126, 203-256 (2015).
  4. Balog, C. I. A. et al. N-glycosylation of colorectal cancer tissues a liquid chromatography and mass spectrometry-based investigation. Mol. Cell. Proteomics 11, 571-585 (2012).
  5. Holst, S. et al. Investigations on aberrant glycosylation of glycosphingolipids in colorectal cancer tissues using liquid chromatography and matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS). Mol. Cell. Proteomics 12, 3081-3093 (2013).
  6. Holst, S. et al. N-glycosylation Profiling of Colorectal Cancer Cell Lines Reveals Association of Fucosylation with Differentiation and Caudal Type Homebox 1 (CDX1)/Villin mRNA Expression. Mol. Cell. Proteomics 15, 124-140 (2016).
  7. Holst, S. et al. Linkage-specific in-situ sialic acid derivatization for N-glycan mass spectrometry imaging of FFPE tissues. Anal. Chem. 88, 5904-5913 (2016).
  8. Heijs, B. et al. Multimodal Mass Spectrometry Imaging of N-glycans and Proteins from the Same Tissue Section. Anal. Chem. 88, 7745-7753 (2016).