Accumulation of somatic mutations is necessary for oncogenic transformation. Somatic mutation patterns in cancer fall into 30 distinct “signatures” based on combinations of single nucleotide variants in the trinucleotide context. The aim of this project is to explore the mechanisms and aetiology underlying COSMIC Signature 17 (Sig17) and impact on cancer development. Sig17 is present in 80% of eosophageal adenocarcinoma (EAC) and 40% gastric adenocarcinoma (GAC). It is characterised by high frequency of T>G mutations which occurs when oxidised guanine (oxoG) from the dNTP pool erroneously pairs with adenine. Bile reflux is a risk factor for EAC and causes oxidative stress and so we hypothesise bile causes Sig17. Interestingly, Sig17 mutations also form hotspots in motifs of CCCTC-binding factor (CTCF) binding sites which are important for transcriptional repression and DNA loop formation. Therefore, Sig17 mutations may drive cancer through altered DNA conformation and long-range gene expression.
Analysing COSMIC Cell Lines Project data revealed the gastric adenocarcinoma cell line AGS as having strongest Sig17 profile. We performed whole genome sequencing on AGS cells at 30X to confirm a weighted Sig17 profile of 23%. Overlapping our AGS mutation calls with CTCF motifs revealed 34 hetereozygous mutations in CTCF binding sites. We subsequently performed CTCF ChIP-seq with allele-specific analysis to confirm that mutant alleles cause loss of CTCF binding. We are currently performing allele-specific capture Hi-C to investigate whether loss of CTCF binding results in changes to DNA loop formation and plan to integrate these findings with allele-specific RNA-seq data to determine if altered loop formation causes oncogenic gene expression. To explore how oxo-guanine gets incorporated into DNA we are utilising an oxoG specific antibody to immunoprecipitate DNA followed by sequencing.
Understanding the aetiology and biological consequences of Sig17 is important for development of preventative strategies and targeted drug selection in these cancers.