Identification of O-Linked Glycoproteins Binding to the Lectin Helix pomatia Agglutinin as Markers of Metastatic Colorectal Cancer.
BACKGROUND
Protein glycosylation is an important post-translational modification shown to be altered in all tumour types studied to date. Mucin glycoproteins have been established as important carriers of O-linked glycans but other glycoproteins exhibiting altered glycosylation repertoires have yet to be identified but offer potential as biomarkers for metastatic cancer.


METHODS
In this study a glycoproteomic approach was used to identify glycoproteins exhibiting alterations in glycosylation in colorectal cancer and to evaluate the changes in O-linked glycosylation in the context of the p53 and KRAS (codon 12/13) mutation status. Affinity purification with the carbohydrate binding protein from Helix pomatia agglutinin (HPA) was coupled to 2-dimensional gel electrophoresis with mass spectrometry to enable the identification of low abundance O-linked glycoproteins from human colorectal cancer specimens.


RESULTS
Aberrant O-linked glycosylation was observed to be an early event that occurred irrespective of the p53 and KRAS status and correlating with metastatic colorectal cancer. Affinity purification using the lectin HPA followed by proteomic analysis revealed annexin 4, annexin 5 and CLCA1 to be increased in the metastatic colorectal cancer specimens. The results were validated using a further independent set of specimens and this showed a significant association between the staining score for annexin 4 and HPA and the time to metastasis; independently (annexin A4: Chi square 11.45, P = 0.0007; HPA: Chi square 9.065, P = 0.0026) and in combination (annexin 4 and HPA combined: Chi square 13.47; P = 0.0002).


CONCLUSIONS
Glycoproteins showing changes in O-linked glycosylation in metastatic colorectal cancer have been identified. The glycosylation changes were independent of p53 and KRAS status. These proteins offer potential for further exploration as biomarkers and potential targets for metastatic colorectal cancer.
Department of Biomedical Sciences, Faculty of Science and Technology, University of Westminster, London, United Kingdom.
Diluka
D
Peiris
Universite des Antilles et de la Guyane, Département Scientifique Interfacultaire, EA929 AIHP-GEODE (BIOSPHERES), Campus de Schœlcher, Martinique.
Marlène
M
Ossondo
Department of Biomedical Sciences, Faculty of Science and Technology, University of Westminster, London, United Kingdom.
Simon
S
Fry
Division of Surgery and Interventional Science, University College London School of Life and Medical Sciences, Royal Free Campus, Pond Street, London, United Kingdom.
Marilena
M
Loizidou
Universite des Antilles et de la Guyane, Département Scientifique Interfacultaire, EA929 AIHP-GEODE (BIOSPHERES), Campus de Schœlcher, Martinique.
Juliette
J
Smith-Ravin
Department of Biomedical Sciences, Faculty of Science and Technology, University of Westminster, London, United Kingdom.
Miriam V
MV
Dwek
D014408:Biomarkers, Tumor / Q000378:metabolism*; D015179:Colorectal Neoplasms / Q000378:metabolism* / Q000473:pathology*; D004252:DNA Mutational Analysis; D015180:Electrophoresis, Gel, Two-Dimensional; D006023:Glycoproteins / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D007150:Immunohistochemistry; D053208:Kaplan-Meier Estimate; D037102:Lectins / Q000378:metabolism*; D009362:Neoplasm Metastasis; D011485:Protein Binding; D040901:Proteomics; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
D014408:Biomarkers, Tumor; D006023:Glycoproteins; C038095:Helix lectin; C117307:KRAS protein, human; D037102:Lectins; D016159:Tumor Suppressor Protein p53; D016283:Proto-Oncogene Proteins p21(ras)