Estrogen Drives Cellular Transformation and Mutagenesis in Cells Expressing the Breast Cancer–Associated R438W DNA Polymerase Lambda Protein

Abstract

Repair of DNA damage is critical for maintaining the genomic integrity of cells. DNA polymerase lambda (POLL/Pol λ) is suggested to function in base excision repair (BER) and non-homologous end-joining (NHEJ), and is likely to play a role in damage tolerance at the replication fork. Here, using next-generation sequencing, it was discovered that the POLL rs3730477 single nucleotide polymorphism (SNP) encoding R438W Pol λ was significantly enriched in the germlines of breast cancer patients. Expression of R438W Pol λ in human breast epithelial cells induces cellular transformation and chromosomal aberrations. The role of estrogen was assessed as it is commonly used in hormone replacement therapies and is a known breast cancer risk factor. Interestingly, the combination of estrogen treatment and the expression of the R438W Pol λ SNP drastically accelerated the rate of transformation. Estrogen exposure produces 8-oxoguanine lesions that persist in cells expressing R438W Pol λ compared to WT Pol λexpressing cells. Unlike WT Pol λ, which performs error-free bypass of 8-oxoguanine lesions, expression of R438W Pol λ leads to an increase in mutagenesis and replicative stress in cells treated with estrogen. Together, these data suggest that individuals who carry the rs3730477 POLL germline variant have an increased risk of estrogen-associated breast cancer.

Mismatch repair single nucleotide polymorphisms and thyroid cancer susceptibility

“…Oestrogen has also been suggested to give rise to cancer-initiating mutations through the formation of DNA adducts and other oxidative lesions, high levels of which have been observed in women with breast, thyroid or ovarian cancer (68). On the other hand, MSH6 is increasingly being implicated in such oestrogen-associated cancers as 1) in vitro oestrogen exposure after catechol-O-methyltransferase inhibition increases the levels of 8-oxo-dG (69), an oxidative DNA lesion whose repair involves the MutSα complex; 2) MSH6 mutations and reduced MSH6 mRNA expression have been reported in breast cancer patients and breast tumour derived cell lines, respectively (70); 3) in LS patients, endometrial cancer is commonly associated with MSH6 mutations (27,31,39,62); 4) MSH2-the binding partner of MSH6 in MutSα-is able to transactivate the oestrogen receptor α, through its MSH6 interaction domain (71); 5) several DNA repair SNPs have been associated with increased oestrogen sensitivity in the development of breast cancer (72)(73)(74). Also, we previously reported a non-significant breast cancer risk increase in rs1042821 variant allele homozygotes (53), in line with the results reported here.…”

The NEIL1 G83D germline DNA glycosylase variant induces genomic instability and cellular transformation

“…Next, to determine if replication stress is present at higher levels in G83D-expressing cells, we performed DNA fiber assays as we previously described [ 23 ] [ 24 ], in the absence and presence of H 2 O 2 , which was used to induce oxidative base damage. We first labeled the DNA of replicating cells for 30 minutes with IdU (red), and then treated with H 2 O 2 to induce oxidative DNA damage (Figure 2A ).…”

Base Excision Repair Variants in Cancer

“…For cellular characterization of BER variants, the cDNA of the variant is stably expressed in immortal but non-transformed human cells. We have experience using the MCF10A or HME-1 mammary epithelial cells [for example see (Galick et al, 2013; Nemec, Bush, Towle-Weicksel, Taylor, Schulz, Weidhaas et al, 2016)]. Here we will describe our work with MCF10A, which is a non-transformed, immortalized mammary epithelial cell line that is near-diploid with a stable karyotype.…”

“…Aberrant BER can occur as a result of deficient removal of damaged bases by DNA glycosylases, inefficient end remodeling or backbone incision by enzymes including PNKP and APE1, slow, defective, or error-prone gap filling by Pol ß, defective scaffolding of the BER enzyme complex by XRCC1, or inefficient ligation. Each of these events has the potential to result in point mutations in growth control genes [for an example see (Donigan, Hile, et al, 2012)] and/or genomic instability [for an example see (Nemec et al, 2016)], but the cells must replicate and divide in order for the phenotype to manifest itself.…”

“…For instance, if expression of a BER variant increases the levels of chromosomal aberrations and micronuclei, as determined by the studies in the previous section, then one possibility is that the BER variant does not remove polymerase-blocking lesions from DNA thereby disrupting replication. Disruption of replication can be tested indirectly by measuring DNA double-strand breaks (DSBs) that arise during S-phase as a result of collapsed replication forks and directly by measuring the initiation and progression of sites of replication using the DNA fiber assay [for an example see (Nemec et al, 2016)]. A phosphorylated form of the histone variant H2A.X (γH2A.X) is used as a marker to measure DSBs and propidium iodide (PI) is used to measure DNA content to delineate G1, S and G2/M phases of the cell cycle by flow cytometry.…”