Colorectal cancer risk variants in the 11q13.4 locus are associated with variable POLD3 transcript expression which may promote DNA damage and telomere shortening in colorectal cancer cells

DNA Polymerase Delta (Pol {delta}) acts in DNA synthesis and in DNA repair, both important factors in the proliferative nature, longevity, and mutability of cancer cells. The chromosomal region containing POLD3, the third subunit of Pol {delta}, has recently been identified by genome wide-association studies as containing single nucleotide polymorphisms (SNPs) that increase the risk of colorectal cancer (CRC). Here, we have used genetic fine mapping and functional annotation tools to identify a haplotype block of 26 SNPS across the POLD3 locus which form a splicing quantitative trait locus associated with context dependent expression of specific POLD3 transcripts. In normal colonic tissue, risk alleles at these SNPs correlate with differential expression of specific POLD3 transcripts, and this pattern appears to be remodelled in colorectal tumours. To explore these statistical associations further we generated CRC cell line models with reduced POLD3 expression levels, to predict how genetic alterations in the POLD3 gene may affect the development of CRC. Knockdown of POLD3 results in reduced proliferative ability and stalling of the cell cycle in S phase, with some apoptosis of CRC cells. Importantly, POLD3 knockdown cells exhibit DNA damage, manifested by double-stranded breaks (DSBs), and upregulation of DSB repair pathways. Reduced POLD3 also correlates with shortened telomeres with disruption of, specifically, the ALT telomeric maintenance pathway. Taken together these observations suggest that low POLD3 expression can increase genetic instability in the form of DSBs and shortened telomeres. This is supported by the observation that low POLD3 expression is associated with reduced overall survival in a sub-set of CRC patients with microsatellite instability, cancers in which DNA repair is already compromised. Author SummaryCommon variations in DNA in the 11q13.4 region on chromosome 11 have been associated with an increased risk of colorectal cancer. Here, we find that a block of 26 of these variants, encompassing the POLD3 genomic region, are associated with changes in splicing and expression of specific POLD3 transcripts making it a likely target gene. We have therefore modelled the effect of reducing expression of POLD3, known for its role in DNA replication and repair, in colorectal cancer cells. We find that, while they grow more slowly, they exhibit much higher DNA damage as well as shorter telomeres. Since both these traits contribute to genomic instability, an enabling characteristic of many cancers, we propose that a modest reduction in POLD3, such as that associated with variants in the 11q13.4 region could explain the associated risk of colorectal cancer. In support of this, overall survival of patients with genetically unstable cancers, is reduced in patients with low POLD3 expression.