Silencing of the EPHB3 tumor-suppressor gene in human colorectal cancer through decommissioning of a transcriptional enhancer.

The protein tyrosine kinase Ephrin type-B receptor 3 (EPHB3) counteracts tumor-cell dissemination by regulating intercellular adhesion and repulsion and acts as tumor/invasion suppressor in colorectal cancer. This protective mechanism frequently collapses at the adenoma-carcinoma transition due to EPHB3 transcriptional silencing. Here, we identify a transcriptional enhancer at the EPHB3 gene that integrates input from the intestinal stem-cell regulator achaete-scute family basic helix-loop-helix transcription factor 2 (ASCL2), Wnt/ß-catenin, MAP kinase, and Notch signaling. EPHB3 enhancer activity is highly variable in colorectal carcinoma cells and precisely reflects EPHB3 expression states, suggesting that enhancer dysfunction underlies EPHB3 silencing. Interestingly, low Notch activity parallels reduced EPHB3 expression in colorectal carcinoma cell lines and poorly differentiated tumor-tissue specimens. Restoring Notch activity reestablished enhancer function and EPHB3 expression. Although essential for intestinal stem-cell maintenance and adenoma formation, Notch activity seems dispensable in colorectal carcinomas. Notch activation even promoted growth arrest and apoptosis of colorectal carcinoma cells, attenuated their self-renewal capacity in vitro, and blocked tumor growth in vivo. Higher levels of Notch activity also correlated with longer disease-free survival of colorectal cancer patients. In summary, our results uncover enhancer decommissioning as a mechanism for transcriptional silencing of the EPHB3 tumor suppressor and argue for an antitumorigenic function of Notch signaling in advanced colorectal cancer.

Class I and III HDACs and loss of active chromatin features contribute to epigenetic silencing of CDX1 and EPHB tumor suppressor genes in colorectal cancer.

Aberrant Wnt/ß-catenin signaling is a driving force during initiation and progression of colorectal cancer. Yet, the Wnt/ß-catenin targets CDX1, EPHB2, EPHB3 and EPHB4 (EPHB2-4) act as tumor suppressors in intestinal epithelial cells and frequently appear to be transcriptionally silenced in carcinomas. The molecular mechanisms which underlie the apparent loss of expression of a subset of Wnt/ß-catenin targets in a background of persistent pathway activity are largely unknown. To gain insight into this, we quantified expression of CDX1 and EPHB2-4 in human tissue specimens of case-matched colorectal normal mucosa, adenoma and invasive carcinoma. In particular EPHB2-4 display biphasic, albeit not strictly coincident, expression profiles with elevated levels in adenomas and decreased transcription in approximately 30% of the corresponding carcinomas. Consistent with their divergent and variable expression we observed considerable heterogeneity among the epigenetic landscapes at CDX1 and EPHB2-4 in a model of colorectal carcinoma cell lines. Unlike the inactive CDX1 locus, EPHB2-4 maintain DNA hypomethylation of their promoter regions in the silent state. A strong reduction of active histone modifications consistently parallels reduced expression of CDX1 and EPHB3 and to some extent of EPHB2. Accordingly, treatment with inhibitors for DNA methyltransferases (DNMTs) and histone deacetylases (HDACs) restored CDX1 and EPHB2-4 expression depending upon epigenetic features at their promoters but also upon cellular background. Overall our findings show that downregulation of CDX1 and EphB receptor genes occurs independently and that different branches of epigenetic control systems including class I and III HDACs contribute to epigenetic silencing of Wnt/ß-catenin targets during colorectal tumorigenesis.