The DNA binding proteins and transcription factors TCF4 and LEF-1 are partners of nuclear β-catenin and effectors of the Wnt/β-catenin signalling pathway, which is decisively involved in tumorigenesis and progression of colorectal cancer. TCF4 is present in the base of the normal colonic crypt where the TCF4/β-catenin complex controls stem cells[17
]. In colorectal cancer the expression of TCF4 as well as LEF-1 has been described [14
], but was not accurately evaluated and compared with nuclear β-catenin positivity.
The present study establishes that the nuclear expressions of TCF4, LEF-1 and β-catenin do not correlate with each other and that TCF4 and LEF-1 positivity is not mutually exclusive in colorectal cancer. In accordance with published literature we found nuclear β-catenin positivity in 75% of cases. In contrast LEF-1 expression was found only in 26% and TCF4 in 46% of colorectal carcinomas. Comparing LEF-1, TCF4 and β-catenin expression, there were cases without nuclear β-catenin which were positive for LEF-1, TCF4 or both factors. Additionally, other cases showed β-catenin positivity, but lacked LEF-1 and TCF4 expression. These findings suggest that activation of the Wnt signalling pathway as indicated by the presence of nuclear β-catenin staining, is not necessarily accompanied by TCF4 or LEF-1 expression. Furthermore, TCF4 and LEF-1 positivity is not restricted to β-catenin positive cases implicating the presence of Wnt-signalling-independent mechanisms, which can additionally regulate the expression of both factors in vivo.
As LEF-1 has been shown to be a target of TCF4/β-catenin [14
], we speculated that tumour progression may be accompanied by a shift of β-catenin binding partners from TCF4 to LEF1 and we therefore expected to find TCF4 positivity mainly in central tumour areas and LEF-1 mainly in the front of invasion. This assumption seemed to be in accordance with studies showing that LEF-1 enhances tumour cell invasiveness [10
] and induces an epithelial to mesenchymal transition [11
]. However in most tumours the expression of these factors was heterogeneously distributed throughout the tumours without a discernable expression pattern. Furthermore when correlating both factors with survival we found that only TCF4 expression was associated with a significant lower overall survival, which fits with the continuous activation of the Wnt/β-catenin signalling pathway in colorectal tumorigenesis and malignant tumour progression [1
]. In contrast LEF-1 expression and the LEF-1/TFC4 coefficient correlated with a significant better overall survival. These surprising findings suggest that TCF4 might be the main binding partner for β-catenin during development and progression of colorectal cancer whereas an enhancement of Wnt/β-catenin transcriptional activity by a switch from TCF4 to LEF-1 is unlikely. Moreover, LEF-1 expression is independent from the TCF4/β-catenin expression.
In fact, LEF-1 expression has been shown to be independently of the canonical Wnt signalling activated by the TGF-β/Smad signalling pathway [22
]. Inhibition of TGFβ signalling plays a role in tumour progression of colorectal cancer [23
] and inactivating mutations of the TGFβ pathway have been shown to cause an induction of growth arrest, differentiation and apoptosis being crucial events during the cancer progression [2
]. Loss of TGF-β responsiveness promotes tumour progression in human colorectal cancers [27
] and overexpression of the TGFβ inhibitor BAMBI causes colon cancer cells to form tumours that metastasize more frequently to liver and lymph nodes than control cancer cells in mural models [28
]. In our study LEF-1 expression in colorectal cancer correlated with an improved patient survival. Therefore LEF-1 expression might indicate an activated TGFβ signalling which reduces tumour progression and development of metastasis.
TCF4 and LEF-1 expression was found to be heterogeneously distributed throughout the tumours, which is in support with the fact that individual tumours are organized hierarchically. Tumors display distinct sub-areas of proliferation, cell-cycle arrest, epithelial differentiation, cell adhesion and dissemination and contain different cell sup-populations like more differentiated tumor cells and tumorigenic cancer stem-like cells (CSC). CSCs are characterized by an activated Wnt/β-catenin signalling pathway [29
] which is indicated by the nuclear expression of β-catenin, and EMT [30
]. Dedifferentiated tumor cells with signs of EMT and nuclear expression of β-catenin which might be CSCs are found at the invasion front of colorectal cancers [31
]. As LEF1 expression was found more often in main tumour areas and correlated with better survival it might indicate differentiated tumor cells without invasive or metastatic potential. In contrast TCF4 expression might indicate cells with traits of CSCs consistent with its function to maintain crypt stem cells of gut epithelium and its correlation with lower survival.