Increased mitochondrial DNA (mtDNA) copy number in peripheral blood cells (PBC) has been associated with the risk of developing several tumor types. Here we evaluate sources of variation of this biomarker, and its association with breast cancer risk in a prospective cohort study. mtDNA copy number was measured using quantitative real-time PCR on PBC DNA samples from participants in the UK-based Breakthrough Generations Study. Temporal and assay variation was evaluated in a serial study of 91 women with two blood samples collected approximately 6 years apart. Then, associations with breast cancer risk factors and risk were evaluated in 1,108 cases and 1,099 controls using a nested case-control design. In the serial study, mtDNA copy number showed low assay variation but large temporal variation (assay intraclass correlation coefficient (ICC)= 79.3%-87.9%; temporal ICC=38.3%). Higher mtDNA copy number was significantly associated with younger age at blood collection, being pre-menopausal, having an older age at menopause and never taking HRT, both in cases and controls. Based on measurements in a single blood sample taken on average 6 years before diagnosis, higher mtDNA copy number was associated with increased breast cancer risk (OR (95%CI) for highest versus lowest quartile = 1.37 (1.02-1.83), P-trend=0.007). In conclusion, mtDNA copy number is associated with breast cancer risk, and represents a promising biomarker for risk assessment. The relatively large temporal variation should be taken into account in future analyses.

TET2 enzymatically converts 5-methylcytosine to 5-hydroxymethylcytosine as well as other covalently modified cytosines and its mutations are common in myeloid leukemia. However, the exact mechanism and the extent to which TET2 mutations affect DNA methylation remain in question. Here, we report on DNA methylomes in TET2 wild-type (TET2-WT) and mutant (TET2-MT) cases of chronic myelomonocytic leukemia (CMML). We analyzed 85,134 CpG sites [28,114 sites in CpG islands (CGI) and 57,020 in non-CpG islands (NCGI)]. TET2 mutations do not explain genome-wide differences in DNA methylation in CMML, and we found few and inconsistent differences at CGIs between TET2-WT and TET2-MT cases. In contrast, we identified 409 (0.71%) TET2-specific differentially methylated CpGs (tet2-DMCs) in NCGIs, 86% of which were hypermethylated in TET2-MT cases, suggesting a strikingly different biology of the effects of TET2 mutations at CGIs and NCGIs. DNA methylation of tet2-DMCs at promoters and nonpromoters repressed gene expression. Tet2-DMCs showed significant enrichment at hematopoietic-specific enhancers marked by H3K4me1 and at binding sites for the transcription factor p300. Tet2-DMCs showed significantly lower 5-hydroxymethylcytosine in TET2-MT cases. We conclude that leukemia-associated TET2 mutations affect DNA methylation at NCGI regions containing hematopoietic-specific enhancers and transcription factor–binding sites. Cancer Res; 75(14); 1–11. ©2015 AACR.

TET2 enzymatically converts 5-methylcytosine to 5-hydroxymethylcytosine as well as other covalently-modified cytosines and its mutations are common in myeloid leukemia. However, the exact mechanism and the extent to which TET2 mutations affect DNA methylation remain in question. Here we report on DNA methylomes in TET2 wild type (TET2-WT) and mutant (TET2-MT) cases of chronic myelomonocytic leukemia (CMML). We analyzed 85,134 CpG sites (28,114 sites in CpG islands (CGIs) and 57,020 in non-CpG islands (NCGIs)). TET2 mutations do not explain genome-wide differences in DNA methylation in CMML, and we found few and inconsistent differences at CGIs between TET2-WT and TET2-MT cases. By contrast, we identified 409 (0.71%) TET2-specific differentially methylated CpGs (tet2-DMCs) in NCGIs, 86% of which were hypermethylated in TET2-MT cases, suggesting a strikingly different biology of the effects of TET2 mutations at CGIs and NCGIs. DNA methylation of tet2-DMCs at promoters and non-promoters repressed gene expression. Tet2-DMCs showed significant enrichment at hematopoietic-specific enhancers marked by H3K4me1, and at binding sites for the transcription factor p300. Tet2-DMCs showed significantly lower 5-hydroxymethylcytosine in TET2-MT cases. We conclude that leukemia-associated TET2 mutations affect DNA methylation at NCGI regions containing hematopoietic-specific enhancers and transcription factor binding sites.

Cancer presents a serious threat to human health. The understanding of the cell fate determination during development and tumor-genesis remains challenging in current cancer biology. It was suggested that cancer stem cell (CSC) may arise from normal stem cells or be transformed from normal differentiated cells. This gives hints on the connection between cancer and development. However, the molecular mechanisms of these cell-type transitions and the CSC formation remain elusive. We quantified landscape, dominant paths, and switching rates between cell types from a core gene regulatory network for cancer and development. Stem cell, CSC, cancer, and normal cell types emerge as basins of attraction on associated landscape. The dominant paths quantify the transition processes among CSC, stem cell, normal cell, and cancer cell attractors. Transition actions of the dominant paths are shown to be closely related to switching rates between cell types, but not always to the barriers in between, because of the presence of the curl flux. During the process of P53 gene activation, landscape topography changes gradually from a CSC attractor to a normal cell attractor. This confirms the roles of P53 of preventing the formation of CSC through suppressing self-renewal and inducing differentiation. By global sensitivity analysis according to landscape topography and action, we identified key regulations determining cell-type switchings and suggested testable predictions. From landscape view, the emergence of the CSCs and the associated switching to other cell types are the results of underlying interactions among cancer and developmental marker genes. This indicates that the cancer and development are intimately connected. This landscape and flux theoretical framework provides a quantitative way to understand the underlying mechanisms of CSC formation and interplay between cancer and development. Cancer Res; 75(13); 1–12. ©2015 AACR.

Cancer presents a serious threat to human health. The understanding of the cell fate determination during development and tumor genesis remains challenging in current cancer biology. It was suggested that cancer stem cell (CSC) may arise from normal stem cells, or be transformed from normal differentiated cells. This gives hints on the connection between cancer and development. However, the molecular mechanisms of these cell type transitions and the CSC formation remain elusive. We quantified landscape, dominant paths and switching rates between cell types from a core gene regulatory network for cancer and development. Stem cell, CSC, cancer, and normal cell types emerge as basins of attraction on associated landscape. The dominant paths quantify the transition processes among CSC, stem cell, normal cell and cancer cell attractors. Transition actions of the dominant paths are shown to be closely related to switching rates between cell types, but not always to the barriers in between, due to the presence of the curl flux. During the process of P53 gene activation, landscape topography changes gradually from a CSC attractor to a normal cell attractor. This confirms the roles of P53 of preventing the formation of CSC, through suppressing self-renewal and inducing differentiation. By global sensitivity analysis according to landscape topography and action, we identified key regulations determining cell type switchings and suggested testable predictions. From landscape view, the emergence of the CSCs and the associated switching to other cell types are the results of underlying interactions among cancer and developmental marker genes. This indicates that the cancer and development are intimately connected. This landscape and flux theoretical framework provides a quantitative way to understand the underlying mechanisms of CSC formation and interplay between cancer and development. Major Findings: We developed a landscape and path theoretical framework to investigate the global natures and dynamics for a core cancer stem cell gene network. Landscape exhibits four basins of attraction, representing CSC, stem cell, cancer and normal cell states. We quantified the kinetic rate and paths between different attractor states. We uncovered certain key genes and regulations responsible for determining the switching between different states.