Abstract P6-07-11: On the origin of T>C transition mutations in breast cancer

Abstract

Abstract Background: Mutations in cancer driver genes are thought to occur early in tumorigenesis. Examination of the sequence context enables the inference of causality. For example, C>A transversions in lung malignancies are ascribed to tobacco smoke. A large proportion of clonal and subclonal mutations in breast cancer are attributed to the APOBEC cytidine deaminases. However, 26% of clonal mutations in cancer genes in the breast and 15% of subclonal mutations are T>C transitions [Sci Transl Med. 2015;7:283ra54]. The purpose of this study was to determine if the T>C transitions are likely an early event in breast carcinogenesis and look for clues to the mechanism by which they are produced. Methods: Histologically normal tissue adjacent to a cancer provides a window into the early steps of oncogenesis. BAM files of exome sequencing data from 11 matched trios of tumor, adjacent normal tissue and blood (leukocyte DNA) from the TCGA (The Cancer Genome Atlas) breast cancer dataset were downloaded from the UCSC cgHUB repository. Mutations were called using the MuTect software (Broad Institute, Cambridge, MA). As a validation cohort, data was downloaded from 29 additional specimens that had been added to TCGA subsequent to the initial data set, and analyzed as above. RNA-Seq data from the initial adjacent normal was downloaded from the TCGA data portal. The epithelia from 20 frozen tissue cores from healthy premenopausal donors to the Susan G. Komen Tissue Bank were microdissected and the RNA isolated. RNA-sequencing was carried out using the Life Technologies SOLiD Platform. RPKM gene expression values from TCGA and sequencing of the Komen normal tissues were merged, quantile normalized, and batch effect corrected. Normalization and differential gene expression was performed using Partek Genomics Suite. Results: C>T and T>C transition mutations make up the majority of the mutations in histologically normal tissue adjacent to breast cancer. Displaying mutations in their trinucleotide context, i.e., by the sequence context immediately 5' and 3' to the mutated base revealed that T>C mutations most frequently occur in the 5'-ATG-3', 5'-CTG-3' and 5'-ATA-3' contexts. Analysis of the RNA-Seq data discovered 1821 genes to be differentially expressed (FDR<5%). Relevant to the questions being addressed in this study, the expression of Nitric Oxide Synthase 3, an enzyme that synthesizes nitric oxide from L-arginine, is 4-times greater in adjacent normal (p= 4.55E-03). [Of note, published data reveals no significant contamination of adjacent normal with tumor in the TCGA breast data.] Conclusions: NOS3 expression is enhanced in certain inflammatory environments as well as by estrogen. Nitric oxide induces DNA damage; one mechanism is through the N2O3 pathway. Reaction of DNA with N2O3 leads to the deamination adenine to form hypoxanthine. Thymine-DNA glycosylase (TDG) initiates base excision repair (BER). Talhaoui et al [Nucleic Acids Res. 2014;42:6300-13] have recently shown that TDG excises T when it is paired with hypoxanthine. Downstream in this aberrant BER pathway the abasic site is repaired to a C resulting in the T>C transition. The excision of the T is efficient only in a specific nucleotide context, which is 5'-TpG-3', as was observed in our study. Citation Format: Choi M, Radovich M, Brown C, Clare SE. On the origin of T>C transition mutations in breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-07-11.