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The role of folate, and its synthetic form, folic acid (FA) in cancer development is highly controversial. A number of studies, although not all, have shown that folate status is inversely related to the risk of developing breast cancer, however, recent studies have suggested high levels of FA may increase breast cancer risk. As FA intake has increased over the last decade, here we have used an animal model to directly test the effect of different levels of FA intake during specific periods of the life-course on the regulation of key cancer determining genes and mammary gland structure.
This study was carried out in accordance with the Home Office Animals (Scientific Procedures) Act (1986). Female C57BL/6 mice were fed a modified AIN93M semi-purified diet containing either 0.2mg/kg (0.2x basal daily recommendation (BDR)), 1 mg/kg (1x BDR): 5 mg/kg (5 x BDR) or 20 mg/kg (20 x BDR) FA for a four-week period during either juvenile-pubertal (JP) period or adulthood. After this period all mice were fed an AIN93M supplemented maintenance diet containing 1 mg/kg of FA for four weeks and tissues collected. mRNA expression of OCT-4, BRCA1, EZH2, BMI1 and SUZ12 was measured by quantitative real time qRT-PCR, changes in the transcriptome by Total RNA-seq (25 million reads) and mammary cell differentiation by immunohistochemistry and carnine red staining.
FA supplementation during the juvenile period led to a persistent decrease in OCT-4 expression, while in adulthood FA induced an increase in expression. There was no effect on BRCA1 expression after FA supplementation in the juvenile period but an increase in BRCA1 expression with 5mg/kg FA supplementation in adulthood. FA supplementation during the juvenile period had no effect on the expression of the polycomb genes EZH2, BMI1, or SUZ12, however FA supplementation at 5mg/kg in adulthood led to an increase in EZH2, BMI1, and SUZ12 mRNA expression. The increase in EZH2 mRNA expression was accompanied by an increase in EZH2 protein expression and increased levels of H3K27trimethylation. Comparison of the mammary transcriptome after FA supplementation in the juvenile period or adulthood revealed only nine transcripts commonly affect by FA supplementation during the two time periods. The top pathway enriched by FA supplementation in juvenile period was DNA repair, while in adulthood it was epithelial to mesenchymal transition. FA supplementation during adulthood also led to an increase in the number of proliferating cells, increased expression of GATA3, a luminal cell marker and ductal hyperplasia. Similar changes in OCT-4, BRCA1 and EZH2 expression were also observed in the mammary gland after feeding a FA deficient diet, with the direction of change being dependent again upon the time of exposure.
These studies show that FA intake can induce significant changes in gene expression and cell phenotype within the mammary gland, dependent upon the timing and intake of FA, with both FA deficiency and supplementation increasing the expression of genes that play a key role in mammary cell proliferation, differentiation and ductal growth, key factors implicated in breast cancer risk.
Folic acid intake has increased over the last decade, however, there are concerns that high levels of folic acid particularly during early life may increase breast cancer risk. In this grant, we have used an animal model to directly test the effect of different levels of folic acid intake during specific periods of the life-course on the activity of cancer determining genes and mammary gland structure.
To determine whether folic acid intake during adolescence or adulthood induces long lasting changes in the activity of cancer determining genes and alterations in mammary gland structure.
Mice were fed a deficient, adequate, supplemented or high level of folic acid during a four-week period in adolescence or adulthood. Gene activity of key cancer determining genes and mammary gland structure was assessed after supplementation.
Folic acid intake has long lasting effects on gene activity in the mammary gland. These effects are dependent upon the amount of folic acid intake and the time of supplementation or deficiency.
A modest increase in FA intake during adulthood led to an increase in the activity of a protein called EZH2, which has previously been linked to breast tumours with poor survival rates as it promotes the reprogramming of the mammary cells to a more primitive embryonic state and the spreading of the cancer to other sites within the body. In this study, the increase in EZH2 expression induced by FA supplementation was also accompanied by the re-programming of mammary cells to a less structured and more invasive cell type.
In contrast, FA supplementation during adolescence led to an increase in the activity of genes involved in DNA repair suggesting that FA supplementation during this time period may be protective of cancer risk.
Interestingly very similar changes in gene expression were observed after FA supplementation and deficiency, consistent with findings from recent epidemiological studies, which suggest a U-shaped relationship between folate intake and cancer risk.
The findings here support the recent epidemiological studies which suggest a U-shaped relationship between folate and cancer risk and identify potential mediators of this altered cancer risk. These findings also show that even a modest increase in FA supplementation can induce the reprogramming of mammary cells and increase the activity of genes that promote cancer risk, although the effect of FA is highly dependent upon the time of FA supplementation.