Discovery of Melanotransferrin as a Serological Marker of Colorectal Cancer by Secretome Analysis and Quantitative Proteomics

To
discover serological colorectal cancer (CRC) markers, we analyzed
cell line secretome to gather proteins of higher potential to be secreted
from tissues into circulation. A total of 898 human proteins were
identified, of which 62.2% were predicted to be released or shed from
cells. The identified proteins were compared with tissue proteomes
to find candidate proteins whose expressions were elevated in tumor
tissues compared with normal tissues as revealed by (i) quantitative
proteomic analysis based on cICAT and mTRAQ or (ii) data mining of
immunohistochemical images piled in Human Protein Atlas database.
By applying various stringent criteria, 11 candidate proteins were
selected. Among these, we validated an significant increase (p = 0.0018) of melanotransferrin (TRFM) at the plasma level
of CRC patients through Western blotting, using 130 plasma samples
containing 30 healthy controls, 80 CRC patients, and 20 patients of
other diseases. Finally, we measured the expression level of TRFM
in 325 plasma samples containing 77 healthy controls and 228 CRC patients
(34.6 ± 4.2 ng/mL and 67.0 ± 6.4 ng/mL, p < 0.0001) through ELISA and demonstrated the area under the receiver
operating characteristic curve of 0.723 (p < 0.0001)
with a 92.5% specificity, 48.2% sensitivity, and 95.7% positive predictive
value. Furthermore, unlike CEA and PAI-1, up-regulation of TRFM in
pathological stages I & II groups compared with stages III &
IV groups lead us to expect the use TRFM for early-stage diagnosis
of CRC. In this study, we suggest TRFM as a potential serological
marker for CRC and expect our discovery strategy to help identify
highly cancer-specific and body-fluid-accessible biomarkers.