title: Differential protein expression on the cell surface of colorectal cancer cells associated to tumor metastasis.
abstract: Progression to metastasis is the critical point in colorectal cancer (CRC) survival. However, the proteome associated to CRC metastasis is very poorly understood at the moment. In this study, we used stable isotope labeling by amino acids in cell culture to compare two CRC cell lines: KM12C and KM12SM, representing poorly versus highly metastatic potential, to find and quantify the differences in protein expression, mostly at the cell surface level. After biotinylation followed by affinity purification, membrane proteins were separated by SDS-PAGE and analyzed using nanoflow LC-ESI-LTQ. A total of 291 membrane and membrane-associated proteins were identified with a p value<0.01, from which 60 proteins were found to be differentially expressed by more than 1.5-fold. We identified a number of cell signaling, CDs, integrins and other cell adhesion molecules (cadherin 17, junction plakoglobin (JUP)) among the most deregulated proteins. They were validated by Western blot, confocal microscopy and flow cytometry analysis. Immunohistochemical analysis of paired tumoral samples confirmed that these differentially expressed proteins were also altered in human tumoral tissues. A good correlation with a major abundance in late tumor stages was observed for JUP and 17-beta-hydroxysteroid dehydrogenase type 8 (HSD17B8). Moreover, the combined increase in JUP, occludin and F11 receptor expression together with cadherin 17 expression could suggest a reversion to a more epithelial phenotype in highly metastatic cells. Relevant changes were observed also at the metabolic level in the pentose phosphate pathway and several amino acid transporters. In summary, the identified proteins provide us with a better understanding of the events involved in liver colonization and CRC metastasis.
authors.affiliation: Protein Technology Unit, Biotechnology Programme, Spanish National Cancer Centre (CNIO), Madrid, Spain.
authors.forename: Jose Luis
authors.initials: JL
authors.lastname: Luque-García
authors.forename: Jorge Luis
authors.initials: JL
authors.lastname: Martínez-Torrecuadrada
authors.forename: Carolina
authors.initials: C
authors.lastname: Epifano
authors.forename: Marta
authors.initials: M
authors.lastname: Cañamero
authors.forename: Ingrid
authors.initials: I
authors.lastname: Babel
authors.forename: J Ignacio
authors.initials: JI
authors.lastname: Casal
mesh_terms: D019921:Biotinylation; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism*; D015179:Colorectal Neoplasms / Q000378:metabolism* / Q000473:pathology*; D019295:Computational Biology; D018450:Disease Progression; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D007553:Isotope Labeling; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D018613:Microscopy, Confocal; D009362:Neoplasm Metastasis; D009363:Neoplasm Proteins / Q000145:classification / Q000378:metabolism*; D011485:Protein Binding; D015203:Reproducibility of Results; D046888:Tissue Array Analysis
chemical_list: D008565:Membrane Proteins; D009363:Neoplasm Proteins