The effect of the level and timing of folic acid supplementation during the life course on the regulation of breast cancer determining genes, mammary morphology and implications for genome stability and tissue differentiation

Folic acid (FA) intake has increased over the past decade; however, there are concerns that high levels of FA, particularly during early life, may increase breast cancer risk. Here a murine model has been used to directly test FA intake during specific periods of the life-course on the regulation of key cancer determining genes, Oct-4 and Brca1, polycomb regulator genes Ezh2, Bmi1 and Suz12, mammary gland morphology and protein expression. Female C57BL/6 mice were fed a modified AIN93M semi-purified diet containing either 1 mg/kg (1x basal daily recommendation (BDR): 5 mg/kg (5 x BDR), 20 mg/kg (20 x BDR) or a FA deficient diet containing 0.2 mg/kg (0.2 x BDR) for a 4 week period during either the juvenile-pubertal (JP) period or adulthood (A). After this period mice were fed an AIN93M maintenance diet for 4 or 12 weeks. The 2nd and 3rd thoracic mammary glands were collected and mRNA expression of Brca1, Oct-4, Ezh2, Bmi1 and Suz12 measured by quantitative real time PCR (qRT-PCR). Levels of Ezh2 protein and its target H3K27 (tri-methyl) were quantified by Western Blot. Mammary gland epithelial morphology was assessed using mammary whole mounts. Epithelial tree density, branch length, branch junctions and number of terminal end buds (TEBs) were calculated. Genome wide differential gene expression was investigated using RNA-seq in juvenile or adult mice fed 5 mg/kg FA 4 weeks post supplementation. Mammary Oct-4, Brca1, basal/myoepithelial marker α-smooth muscle actin (α-SMA), luminal marker keratin 8 (K8), cell proliferation marker Ki67 and driver of luminal differentiation Gata-3 proteins were investigated with immunohistochemistry using 3,3’-diaminobenzidine and immunofluorescence. A persistent increase in Brca1, Oct-4, Ezh2, Bmi1 and Suz12 gene expression after FA supplementation at 5 mg/kg was observed during A. In contrast, FA supplementation at 5 mg/kg during JP led to a decrease in Oct-4 gene expression. FA supplementation at 5 mg/kg increased levels of Ezh2 protein and levels of H3K27 (tri-methyl) during A. Mammary morphology was largely unchanged; however, epithelial density and number of TEBs showed a persistent increase in JP supplemented mice. Brca1, and Oct-4 proteins were found to be localised in mammary epithelial ducts. Numbers of basal/myoepithelial cells expressing α-SMA were unchanged, however, numbers of cells expressing cell proliferation marker Ki67 increased. RNA-seq analysis identified differential expression in genes associated with extracellular matrix, enriched gene sets included epithelial mesenchymal transition (EMT) and pathways associated with matrix metalloproteinases (MMPs), extracellular matrix components and cancer during A. Interestingly, juvenile mice showed distinct gene sets with minimal overlap with those during A. JP and adult FA supplementation induce tissue specific alterations in expression of key cancer genes that persist beyond the period of supplementation. The effects of FA are both dose and timing specific and imply that the ability of FA to modify cancer risk extends beyond recent diet and may, in part, reflect intakes during different periods of mammary plasticity. These findings suggest high folic acid intake in mice may increase breast cancer risk especially when consumed during adulthood.