Metastasis remains one of the major challenges in management of CRC patients. LNM is the most common form of metastasis in CRC. To develop LNM-associated biomarkers for CRC, we employed the quantitative proteomic strategy of methyl esterification stable isotope labeling coupled with 2D-LC-MS/MS. A total of 644 proteins were identified, including 43 that were differentially expressed by at least 2.5-fold between LNM CRC and non-LNM CRC. We found many of the 43 proteins that possibly participate in the biological processes associated with tumor metastasis, such as cell motility and adhesion, migration, and signal transduction.
Among the upregulated proteins, annexin A2, one of the calcium- and phospholipid-binding proteins, has been widely reported in various cancers with the regulation of cell growth, motility, invasion and signaling pathways[20
]. The increase in caveolin-1 performs the functions of signal transduction, cell transformation and anti-apoptotic activity[21
]. Moreover, caveolin-1 has been found to be overexpressed in several multidrug-resistant cancer cell lines[22,23
]. In addition, some downregulated proteins identified in our study have also been observed to possess similar biological effects, including galectin-3-binding protein[24
] and cyclin-G-associated kinase[25
Previously, Pei et al[26
] have carried out a proteomic study on 10 CRC samples using conventional 2D electrophoresis coupled with MALDI-TOF-MS, and have reported a pattern of four differentially expressed proteins potentially associated with LNM. In contrast, our results revealed up to 43 proteins that were differentially expressed by at least 2.5-fold. Annexin A2 and glutathione S-transferase P1 both correlated with LNM in our study and that of Pei et al Heat shock protein-27 and liver fatty acid binding protein (L-FABP) were found to correlate with LNM by Pei et al, but not in our study, whereas 41 proteins identified in our study were not listed by Pei et al This discordance is probably due to the different clinical background of the samples included in the studies, and the different proteomic strategies used. It is required to analyze systemically and integrate all the complementary data from various institutions into a common databank to elucidate exactly the molecular background of CRC. In addition, Pang et al[27
] have identified and confirmed six differentially expressed proteins (e- fatty acid binding protein 5, methylcrotonoyl Coenzyme A carboxylase 2, pyrophosphatase 2, synaptotagmin-like protein 2, Ezrin, and smooth muscle protein) that are associated with LNM in prostate cancer by DIGE-based proteome analysis. However, there was no concordance between the results in that study and our study, which is probably mainly due to the different cancers and proteomic approaches included.
Recently, several studies have shown that S100A4 is an important factor relevant to progression and prognosis in various human cancers, such as thyroid[28
] and colorectal[32
] cancer. In particular, several studies have revealed that overexpression of S100A4 strongly indicates the presence of LNM[15-17
], which agrees with our original study aim. However, similar investigations have been limited between S100A4 expression and LNM in CRC. In view of the above reasons, S100A4, one of the significantly upregulated proteins identified in LNM CRC compared with non-LNM CRC, which has been confirmed at the protein and mRNA levels, attracted our attention and interest.
S100A4, also known as 18A2/mts1, CAPL, PEL-98, 42A, p9Ka, and metastasin, belongs to the S100 super-family of calcium-binding proteins[33
]. S100A4-mediated calcium signaling plays a major role in crucial biological functions that influence various aspects of cell physiology, including proliferation and apoptosis, and differentiation and morphogenesis. It is also significantly involved in cell adhesion and motility, and cancer invasion and metastasis[34,35
]. A large body of evidence suggests that S100A4 is involved in cell metastatic phenotype by modulating the cytoskeletal dynamics, cadherin/catenin complex cytoskeletal linkage, CD44/cytoskeletal linkage, and extracellular-matrix-associated proteolytic enzyme. Furthermore, S100A4 can participate in the activation of the matrix metalloproteinase/tissue inhibitor of metalloproteinase system and angiogenic factor vascular endothelial growth factor, which in turn can lead to tumor neovascularization[36,37
To study further the relationship between S100A4 expression and the LNM phenotype of CRC, and determine whether S100A4 could be a prognostic factor in predicting clinical outcomes of CRC patients, we examined an additional 112 archived CRC samples for S100A4 expression. We found that the elevation in S100A4 expression level was significantly correlated with LNM and advanced TNM stage, which suggests that S100A4 plays an important part in the progression of CRC from a localized to lymph node metastatic disease. In addition, patients with S100A4-positive CRC had an increasing risk of recurrence and significantly reduced overall survival. Univariate and multivariate analyses indicated that S100A4 expression is a powerful independent prognostic factor for recurrence and overall survival in CRC, which indicates the considerable prognostic value of S100A4 expression.
In conclusion, our current study employed a quantitative proteome analysis to profile the differently expressed proteins associated with LNM in CRC. S100A4 was identified and confirmed to be significantly overexpressed in LNM CRC. Further evaluation in an independent sample set has suggested that S100A4 acts as a powerful biomarker for LNM and prognosis in CRC. However, many questions remain to be answered with respect to the cellular function of S100A4 and how it exerts its influence on metastatic progression, with further investigations on our part in progress. We also identified a number of proteins besides S100A4 that might provide a more profound insight into the mechanism of LNM in CRC and merit further research.