The Regulation of Potential Driver Genes in Colorectal Cancer

Around 13,000 people die from colorectal cancer (CRC) in England every year. The response rate of combination therapy in patients with advanced disease is around 50%, indicating a need for further therapeutics that are tailored to a patient’s genomic background, with epigenetic treatments fast becoming an emerging field. Two in-house screens combined with bioinformatic analysis of publically available data identified SNF2-related CREBBP Activator Protein (SRCAP) as a potential driver gene of CRC. SRCAP is the core catalytic component of a multi-subunit chromatin-remodeling factor, responsible for the replacement of the H2A histone with its variant H2A.Z within the nucleosome, in order to promote open chromatin and subsequent transcription initiation. We therefore hypothesised that SRCAP is a driver gene of CRC and could provide a novel druggable target. To address this hypothesis, SRCAP levels were either reduced or increased within CRC cells, and several consequential phenotypic hallmarks of cancer determined.

Over-expression of SRCAP in cell lines using cDNA proved challenging to accomplish, due to transfection efficiencies, the size of the construct and a lack of validated antibodies. However, using a CRISPR/dCas9 activation system, over-expression of endogenous SRCAP could be established, which was associated with increased levels of DNA damage. However, due to unstable expression throughout the experimental process, the results were not reproducible and we were not able to draw confident conclusions. Knockdown of SRCAP was successful in two CRC cell lines, and caused an increase in genomic instability, alterations to cellular cycling and decreased growth over time, and an increased sensitivity to clinically used chemotherapeutics in a TP53-dependent manner. SRCAP depletion in an oncogene activated cell background again showed increased DNA damage and cell cycle profile changes but with differing outcomes in different cell lines. Preliminary evidence suggests that SRCAP depletion alone was the main driving force behind these phenotypes. Finally genes associated with the progression of cancer were also identified in whole-genome RNA microarray analyses of SRCAP knockdown and knock up samples.