title: High sensitivity isoelectric focusing to establish a signaling biomarker for the diagnosis of human colorectal cancer.
abstract: The progression of colorectal cancer (CRC) involves recurrent amplifications/mutations in the epidermal growth factor receptor (EGFR) and downstream signal transducers of the Ras pathway, KRAS and BRAF. Whether genetic events predicted to result in increased and constitutive signaling indeed lead to enhanced biological activity is often unclear and, due to technical challenges, unexplored. Here, we investigated proliferative signaling in CRC using a highly sensitive method for protein detection. The aim of the study was to determine whether multiple changes in proliferative signaling in CRC could be combined and exploited as a "complex biomarker" for diagnostic purposes.
  
  
  
  We used robotized capillary isoelectric focusing as well as conventional immunoblotting for the comprehensive analysis of epidermal growth factor receptor signaling pathways converging on extracellular regulated kinase 1/2 (ERK1/2), AKT, phospholipase Cγ1 (PLCγ1) and c-SRC in normal mucosa compared with CRC stage II and IV. Computational analyses were used to test different activity patterns for the analyzed signal transducers.
  
  
  
  Signaling pathways implicated in cell proliferation were differently dysregulated in CRC and, unexpectedly, several were downregulated in disease. Thus, levels of activated ERK1 (pERK1), but not pERK2, decreased in stage II and IV while total ERK1/2 expression remained unaffected. In addition, c-SRC expression was lower in CRC compared with normal tissues and phosphorylation on the activating residue Y418 was not detected. In contrast, PLCγ1 and AKT expression levels were elevated in disease. Immunoblotting of the different signal transducers, run in parallel to capillary isoelectric focusing, showed higher variability and lower sensitivity and resolution. Computational analyses showed that, while individual signaling changes lacked predictive power, using the combination of changes in three signaling components to create a "complex biomarker" allowed with very high accuracy, the correct diagnosis of tissues as either normal or cancerous.
  
  
  
  We present techniques that allow rapid and sensitive determination of cancer signaling that can be used to differentiate colorectal cancer from normal tissue.
authors: Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Dag Hammarskjöldsv 20, Uppsala, 751 85, Sweden.
authors: Narendra
authors: N
authors: Padhan
authors: Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Dag Hammarskjöldsv 20, Uppsala, 751 85, Sweden.|Stockholm Bioinformatics Centre, Science for Life Laboratory, Box 1031, 171 21, Solna, Sweden.|Current address: Department of Mechanical Engineering, National Cheng Kung University, No. 1 University Road, Tainan, 70101, Taiwan.
authors: Torbjörn E M
authors: TE
authors: Nordling
authors: Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Dag Hammarskjöldsv 20, Uppsala, 751 85, Sweden.
authors: Magnus
authors: M
authors: Sundström
authors: Department Surgical Sciences, Uppsala University, 751 85, Uppsala, Sweden.
authors: Peter
authors: P
authors: Åkerud
authors: Department Surgical Sciences, Uppsala University, 751 85, Uppsala, Sweden.
authors: Helgi
authors: H
authors: Birgisson
authors: Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Dag Hammarskjöldsv 20, Uppsala, 751 85, Sweden.
authors: Peter
authors: P
authors: Nygren
authors: Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Dag Hammarskjöldsv 20, Uppsala, 751 85, Sweden.
authors: Sven
authors: S
authors: Nelander
authors: Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Dag Hammarskjöldsv 20, Uppsala, 751 85, Sweden. lena.welsh@igp.uu.se.
authors: Lena
authors: 0000-0003-4275-2000
authors: L
authors: Claesson-Welsh
mesh_terms: D014408:Biomarkers, Tumor; D001706:Biopsy; D000081247:CSK Tyrosine-Protein Kinase; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D015179:Colorectal Neoplasms / Q000175:diagnosis* / Q000235:genetics / Q000378:metabolism*; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007525:Isoelectric Focusing / Q000379:methods; D048052:Mitogen-Activated Protein Kinase 3 / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D009367:Neoplasm Staging; D051966:Phospholipase C gamma / Q000378:metabolism; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000378:metabolism; D012680:Sensitivity and Specificity; D015398:Signal Transduction; D019061:src-Family Kinases / Q000378:metabolism
chemical_list: D014408:Biomarkers, Tumor; D066246:ErbB Receptors; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; C000631346:CSK protein, human; D051057:Proto-Oncogene Proteins c-akt; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D048052:Mitogen-Activated Protein Kinase 3; D051966:Phospholipase C gamma
keywords: Colorectal cancer; ERK; Isoelectric focusing; Proliferation; Signal transduction; c-SRC