Investigating the importance of BCL-3/β-catenin signalling in colorectal tumorigenesis


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Penny Timms1,Danny Legge1,Tracey Collard1,Adam Chambers1,Ann Williams1
1University of Bristol

Abstract

Background

BCL-3, identified as a key regulator of NF-kB signalling, is an important oncogenic player in solid tumours reported to be over-expressed in a subset of colorectal cancers (CRC). We and others have shown that BCL-3 is a potent survival factor in tumour cells. More recently we have demonstrated that BCL-3 acts as a co-activator of β-catenin/TCF-mediated transcription in colorectal cancer cells and that this interaction is important for Wnt-regulated intestinal stem cell gene expression. Given the significance of Wnt/β-catenin signalling in colorectal tumorigenesis, the aim of this study is to use a proteomic approach to further investigate the impact of BCL-3/β-catenin signalling on colorectal tumour progression.

Method

BCL-3 Co-IP experiments were performed in CRC nuclear-enriched lysates. qRT-PCR analysis of Wnt-target gene expression in CRC cells was performed following BCL-3 suppression. CRC lines are grown in Matrigel®, forming spheroids. CRISPR-Cas9D10A based genome editing is performed in CRC cell lines with sgRNAs designed to target exon 4 of human BCL-3. A quantitative proteomic approach using BCL-3 knockout CRC cells and informed by β-catenin transcriptomic data, is used to identify novel targets regulated by the BCL-3/β-catenin complex.

Results

BCL-3 interacts with β-catenin and acts as a co-activator of β-catenin/TCF-mediated transcriptional activity in colorectal cancer cell lines. BCL-3 suppression reduces β-catenin dependant transcription of intestinal stem cell genes LGR5 and ASCL2 and reduces colorectal spheroid and tumoursphere formation. The functional importance of further targets identified are investigated in EMT and spheroid growth assays.

Conclusion

These data suggest BCL-3 acts as a driver for stem cell plasticity in colorectal cancer by pushing cells towards a more stem cell-like phenotype enhancing tumour cell survival and promoting tumour progression. Targeting BCL-3 in those colorectal patients with high expression may represent an exciting new approach by targeting the plasticity of cancer stem cells and improving patient prognosis.