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Abstract
Liver metastasis in colorectal cancer is the major cause of cancer-related deaths. To identify and characterize proteins associated with colon cancer metastasis, we have compared the conditioned serum-free medium of highly metastatic KM12SM colorectal cancer cells with the parental, poorly metastatic KM12C cells using quantitative stable isotope labeling by amino acids in cell culture (SILAC) analyses on a linear ion trap-Orbitrap Velos mass spectrometer. In total, 1337 proteins were simultaneously identified in SILAC forward and reverse experiments. For quantification, 1098 proteins were selected in both experiments, with 155 proteins showing >1.5-fold change. About 52% of these proteins were secreted directly or using alternative secretion pathways. GDF15, S100A8/A9, and SERPINI1 showed capacity to discriminate cancer serum samples from healthy controls using ELISAs. In silico analyses of deregulated proteins in the secretome of metastatic cells showed a major abundance of proteins involved in cell adhesion, migration, and invasion. To characterize the tumorigenic and metastatic properties of some top up- and down-regulated proteins, we used siRNA silencing and antibody blocking. Knockdown expression of NEO1, SERPINI1, and PODXL showed a significant effect on cellular adhesion. Silencing or blocking experiments with SOSTDC1, CTSS, EFNA3, CD137L/TNFSF9, ZG16B, and Midkine caused a significant decrease in migration and invasion of highly metastatic cells. In addition, silencing of SOSTDC1, EFNA3, and CD137L/TNFSF9 reduced liver colonization capacity of KM12SM cells. Finally, the panel of six proteins involved in invasion showed association with poor prognosis and overall survival after dataset analysis of gene alterations. In summary, we have defined a collection of proteins that are relevant for understanding the mechanisms underlying adhesion, migration, invasion, and metastasis in colorectal cancer.
Highlights
Despite the efforts for colorectal cancer (CRC)1 prevention using different strategies [1,2,3,4,5,6], 30 – 40% of patients have regionally advanced disease or suffer from metastasis when diagnosed [7]
Protein Identification and Quantification in KM12 Conditioned Media Using SILAC—Conditioned media from highly metastatic KM12SM and poorly metastatic KM12C cells were investigated for differences in secreted proteins that could be associated with CRC metastasis
GDF15, S100A8/A9, and SERPINI1 Are Candidate Biomarkers for Colorectal Cancer Diagnosis—One of the goals of this study was to identify proteins deregulated in the KM12SM metastatic cell line that could be used in human serum as CRC biomarkers
Summary
Despite the efforts for colorectal cancer (CRC) prevention using different strategies [1,2,3,4,5,6], 30 – 40% of patients have regionally advanced disease or suffer from metastasis when diagnosed [7]. Genetic changes leading to the development of sporadic colorectal cancer primary tumors in intestinal cells have been relatively well characterized [9], further efforts are necessary to better understand the biology of CRC metastasis and to identify associated markers that can be used as diagnostic/. In the case of CRC, there are only three proteins currently used as biomarkers: the carcinoembryonic antigen (CEA) for recurrence and metastasis [1], deleted in colorectal carcinoma (DCC), and vascular endothelial growth factor (VEGF). Recent studies applied iTRAQ or label-free quantification to other pairs of isogenic, nonmetastatic-metastatic colorectal cancer cell lines, SW480 and SW620, for the characterization of protein differences in the whole cell proteome [22] and secretome [23], respectively. KM12SM cells were chosen based on their capacity for liver metastasis, which makes them most appropriate for the study of liver homing and late stages of metastasis
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