Decreased mitochondrial mutagenesis during transformation of breast stem cells into tumorigenic cells

1 Rare stochastic mutations may accumulate during dormancy of stem-like cells, but technical 2 limitations in DNA sequencing have limited exploring this possibility. In this study, we 3 employed a recently established deep sequencing method termed Duplex Sequencing to conduct 4 a genome-wide analysis of mitochondrial (mt) DNA mutations in a human breast stem cell 5 model that recapitulates the sequential stages of breast carcinogenesis. Using this method, we 6 found significant differences in mtDNA amongst normal stem cells, immortal/preneoplastic cells, 7 and tumorigenic cells. Putative cancer stem-like cell (CSC) populations and mtDNA copy 8 numbers increased as normal stem cells become tumorigenic cells. Transformed cells exhibited 9 lower rare mutation frequencies of whole mtDNA than did normal stem cells. The predicted 10 mtDNA rare mutation pathogenicity was significantly lower in tumorigenic cells than normal 11 stem cells. Major rare mutation types in normal stem cells are C>T/G>A and T>C/A>G 12 transitions, while only C>T/G>A are major types in transformed cells. We detected a total of 13 1220 rare point mutations, 678 of which were unreported previously. With only one possible 14 exception (m10342T>C), we did not find specific mutations characterizing mtDNA in human 15 breast CSC; rather, the mitochondrial genome of CSC displayed a decrease in rare mutations 16 overall. Based on our work, we suggest that this decrease (in particular T>C/A>G transitions), 17 rather than the presence of specific mitochondrial mutations, may constitute an early biomarker 18 for breast cancer detection. Our findings support the hypothesis that the mitochondrial genome is 19 altered greatly as a result of the transformation of normal stem cells to CSC, and that mtDNA 20 mutation signatures may aid in delineating normal stem cells from CSC. 21