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. Methodology 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). Conclusion 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.

[1]  Supriya Patel,et al.  Prognostic and Predictive Roles of KRAS Mutation in Colorectal Cancer , 2012, International journal of molecular sciences.

[2]  Anatoliy Markiv,et al.  Beyond the genome and proteome: targeting protein modifications in cancer. , 2012, Current opinion in pharmacology.

[3]  M. Sun,et al.  The association of annexin A2 and cancers , 2012, Clinical and Translational Oncology.

[4]  E. Manor,et al.  O-Linked β-N-Acetylglucosaminylation (O-GlcNAcylation) in Primary and Metastatic Colorectal Cancer Clones and Effect of N-Acetyl-β-d-glucosaminidase Silencing on Cell Phenotype and Transcriptome* , 2012, The Journal of Biological Chemistry.

[5]  Pamela Greenwell,et al.  The lectin Helix pomatia agglutinin recognizes O-GlcNAc containing glycoproteins in human breast cancer. , 2012, Glycobiology.

[6]  M. Dwek,et al.  Cell surface glycan-lectin interactions in tumor metastasis. , 2011, Acta histochemica.

[7]  G. Hart,et al.  Cross talk between O-GlcNAcylation and phosphorylation: roles in signaling, transcription, and chronic disease. , 2011, Annual review of biochemistry.

[8]  Haike Ghazarian,et al.  A glycobiology review: carbohydrates, lectins and implications in cancer therapeutics. , 2011, Acta histochemica.

[9]  U. Schumacher,et al.  Lectin histochemistry of metastasizing and non-metastasizing breast and colon cancer cells. , 2011, Anticancer research.

[10]  L. Gam,et al.  Chemometrics of differentially expressed proteins from colorectal cancer patients. , 2011, World journal of gastroenterology.

[11]  Wengong Yu,et al.  O-GlcNAcylation is a novel regulator of lung and colon cancer malignancy. , 2011, Biochimica et biophysica acta.

[12]  G. Curley,et al.  Identification, Cloning, and Characterization of Two N-Acetylgalactosamine-binding Lectins from the Albumen Gland of Helix pomatia , 2011, The Journal of Biological Chemistry.

[13]  Susan J Fisher,et al.  Sweetening the pot: adding glycosylation to the biomarker discovery equation. , 2010, Clinical chemistry.

[14]  Shuhan Sun,et al.  Expression of Annexin A5 Is Associated With Higher Tumor Stage and Poor Prognosis in Colorectal Adenocarcinomas , 2009, Journal of clinical gastroenterology.

[15]  Pier Giorgio Righetti,et al.  Immobilized pH gradients , 2009, Electrophoresis.

[16]  Xiaobing Chen,et al.  Relationship between expression of CEA, E-cadherin and liver metastasis in colorectal cancer , 2008 .

[17]  J. Casal,et al.  Proteome analysis of membrane fractions in colorectal carcinomas by using 2D-DIGE saturation labeling. , 2008, Journal of proteome research.

[18]  S. Hanash,et al.  Lectin precipitation using phytohemagglutinin‐L4 coupled to avidin–agarose for serological biomarker discovery in colorectal cancer , 2008, Proteomics.

[19]  G. Murray,et al.  Characterisation and protein expression profiling of annexins in colorectal cancer , 2007, British Journal of Cancer.

[20]  M. Dwek,et al.  Proteome analysis of metastatic colorectal cancer cells recognized by the lectin Helix pomatia agglutinin (HPA) , 2007, Proteomics.

[21]  W. Yang,et al.  Modification of p53 with O-linked N-acetylglucosamine regulates p53 activity and stability , 2006, Nature Cell Biology.

[22]  Anne Imberty,et al.  Biochemical and Structural Analysis of Helix pomatia Agglutinin , 2006, Journal of Biological Chemistry.

[23]  Wolfgang Schima,et al.  Liver metastases of colorectal cancer: US, CT or MR? , 2005, Cancer imaging : the official publication of the International Cancer Imaging Society.

[24]  T. Ishikawa,et al.  Clinical significance of serum CEA protein and CEA mRNA after resection of colorectal liver metastases. , 2005, Anticancer research.

[25]  C. Scharf,et al.  Increased expression and altered location of annexin IV in renal clear cell carcinoma: a possible role in tumour dissemination. , 2004, Cancer letters.

[26]  Pier Giorgio Righetti,et al.  Blue silver: A very sensitive colloidal Coomassie G‐250 staining for proteome analysis , 2004, Electrophoresis.

[27]  S. Moss,et al.  The annexins , 2004, Genome Biology.

[28]  R. Elble,et al.  The Breast Cancer β4 Integrin and Endothelial Human CLCA2 Mediate Lung Metastasis* , 2001, The Journal of Biological Chemistry.

[29]  S. Bustin,et al.  Expression of the Ca2+-activated chloride channel genes CLCA1 and CLCA2 is downregulated in human colorectal cancer. , 2001, DNA and cell biology.

[30]  A. Görg,et al.  The current state of two‐dimensional electrophoresis with immobilized pH gradients , 2000, Electrophoresis.

[31]  J. Benz,et al.  Annexins: from structure to function. , 1997, Biological chemistry.

[32]  U. Schumacher,et al.  Is the lectin binding pattern of human breast and colon cancer cells influenced by modulators of sialic acid metabolism? , 1996, Histochemistry and Cell Biology.

[33]  U. Schumacher,et al.  Histochemistry to detect Helix pomatia lectin binding in breast cancer: methodology makes a difference. , 1996, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[34]  U Schumacher,et al.  Helix pomatia agglutinin binding is a useful prognostic indicator in colorectal carcinoma , 1994, Cancer.

[35]  K. Sugimachi,et al.  Prognostic value of the histochemical expression of Helix pomatia agglutinin in advanced colorectal cancer , 1994, Diseases of the colon and rectum.

[36]  A. Leathem,et al.  Prediction of lymph node involvement in breast cancer by detection of altered glycosylation in the primary tumour , 1991, The Lancet.

[37]  S. Tsugane,et al.  Prognostic contributions of Helix pomatia and carcinoembryonic antigen staining using histochemical techniques in breast carcinomas. , 1989, Japanese journal of clinical oncology.

[38]  L. Bhattacharyya,et al.  Lectin-carbohydrate interactions. Studies of the nature of hydrogen bonding between D-galactose and certain D-galactose-specific lectins, and between D-mannose and concanavalin A. , 1988, European journal of biochemistry.

[39]  I. Dokal,et al.  Lectin binding to normal and malignant breast tissue. , 1983, Diagnostic histopathology.

[40]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[41]  J. Turnay,et al.  Differentiation of human colon adenocarcinoma cells alters the expression and intracellular localization of annexins A1, A2, and A5 , 2005, Journal of cellular biochemistry.