In this study, we demonstrated that the combination of the loss of E-cadherin and gain of S100A4 in the invasive margin of CRC is an independent prognostic factor of a poorer outcome, along with AJCC stage and perineural invasion. Nuclear expression of β-catenin was not related to patient survival.
When tumor cells start to invade and metastasize, adhesion molecules undergo alterations. Down-regulation of E-cadherin in CRC is associated with malignant features. Loss of E-cadherin has been shown to be associated with tumor budding  and lymph node metastasis in CRC  and to predict disease recurrence and long-term survival in CRC [8, 20, 21]. In this study, loss of E-cadherin at the invasive margin of CRCs was associated with high tumor budding, perineural invasion, and a poor prognosis.
S100A4 is localized in the nucleus, cytoplasm, and extracellular space and has a wide range of biological functions ranging from regulation of angiogenesis to cell survival, motility, and invasion . We found that S100A4 expression in the invasive margin was increased in infiltrative tumors, those with a lymph node metastasis, advanced AJCC stage, or lymphovascular- and perineural invasion, which are all parameters representing tumor aggressiveness. We demonstrated that high expression of S100A4 is associated with recurrence and mortality. These results were consistent to the previous studies [15, 22, 23], in which S100A4 is related to a poor prognosis. One study reported that S100A4 is overexpressed in cell populations enriched for stem-like cells, which is associated with Wnt/APC/β-catenin signaling pathway and S100A4 worked as β-catenin/TCF target gene . Inconsistently other results, S100A4 was also related to lymphocyte infiltration, which protects tumor progression and destroys tumor budding. These findings were resulted from that S100A4 expressing fibroblasts, monocytes, macrophages, T lymphocytes, neutrophilic granulocytes, or endothelial cells may be misinterpreted as S100A4 expressing cancer cells .
β-catenin plays to maintain cell-to-cell adhesion along with E-cadherin. However, β-catenin also acts as a transcription factor in the Wnt signal transduction pathway and Wnt signaling dysfunction leads to the nuclear accumulation of ß-catenin . Several studies have reported that nuclear ß-catenin expression in the invasive margin is associated with high tumor budding and poor prognosis , whereas other studies did not find such a relationship [9, 12, 20]. In addition, recent study showed that aberrant β-catenin expression was related to favorable prognosis . We did not find any association between β-catenin expression and tumor budding or overall survival in our patient cohort. These inconsistencies may result from different CRC subtypes, the existence of more than one type of CRC in a study , or different evaluation methods . We showed that β-catenin nuclear expression was increased in MSS tumors compared to MSI tumors and that it was higher in left CRCs than right CRCs. These results are consistent with those of a previous study , which reported that MSI-high, CpG island methylator phenotype (CIMP)-high, and BRAF mutations, which are characteristics of right CRCs, showed an inverse association with cytoplasmic and nuclear β-catenin expression. MSS and MSI colorectal cancers are increasingly being recognized as distinct entities with significantly different pathological characteristics, behaviors, and prognoses . MSI is associated with significantly lower levels of nuclear ß-catenin and impaired EMT than MSS . In agreement with these reports, we found that 44.4% of MSI-H cases versus 72.4% of MSS or MSI-L cases were tumor budding positive.
Aberrant expression of E-cadherin, β-catenin, and S100A4 showed parallel patterns each other. These results suggested that overexpression of S100A4 inhibits E-cadherin expression and β-catenin plays a role in driving S100A4-dependent EMT induction . Although individual change of E-cadherin and S100A4 was related to patients’ prognosis in univariate survival analysis, multivariate Cox analysis revealed that the combination of E-cadherin loss and S100A4 overexpression was the only prognostic factor in addition to AJCC stage, which is still the most important prognostic factor in CRC. These results highlight the fact that multi-marker phenotypes rather than single protein are needed in IHC biomarker investigations .
Because the tumor biology of the invasive margin is different than that of the tumor center, the patterns of EMT protein expression are expected to be different at the two sites. In this study, EMT-related changes in the expression of E-cadherin, β-catenin and S100A4 were more severe in the invasive margin than in the tumor center and EMT changes in the invasive margin, but not the tumor center, had prognostic significance.
Tumor budding is a putative hallmark of CRC cell invasion and has previously been shown to be associated with various clinicopathological parameters, including lymph node metastasis, vascular and lymphatic invasion, distant metastasis, local recurrence, and poor outcomes . In this study, high tumor budding was associated with the ratio of metastatic lymph nodes, type of tumor growth, and perineural invasion (data not shown). It has also been classified as an additional prognostic factor. However, our results suggest that the combination of E-cadherin and S100A4 expression at the invasive margin of CRC is superior to tumor budding for predicting prognosis.
The mRNA levels of E-cadherin, β-catenin, and S100A4 were not found to be associated with histopathological parameters or prognosis in this study. For β-catenin and S100A4, mRNA levels reflected that of the encoded protein, but this was not the case for E-cadherin. However, the switch in expression from E-cadherin to N-cadherin and the higher expression of N-cadherin in patients with a poor prognosis were demonstrated in this study. Recent study also showed that N-cadherin was highly expressed in type II papillary renal cell carcinomas than type I cancers and type II cancers were related to poor prognosis .
In conclusion, our results suggest that aberrant expression of E-cadherin and S100A4 in the invasive margin was well related with clinicopathological parameters and IHC of both proteins is useful marker to predict prognosis in CRC.