Folate, DNA Methylation, and Breast Cancer Metastasis

Institution: University of California, Davis
Investigator(s): Teresa Marple, Ph.D. -
Award Cycle: 2008 (Cycle 14) Grant #: 14FB-0135 Award: $78,647
Award Type: Postdoctoral Fellowship
Research Priorities
Etiology and Prevention>Etiology: the role of environment and lifestyle

Initial Award Abstract (2008)

Folate is a B vitamin that is essential for making DNA and controlling gene expression. Both normal cells and cancer cells need folate for these purposes. In 1998, the United States Government mandated cereal and grain fortification with folic acid (the supplement form of folate) to reduce the number of birth defects. The folic acid fortification program has been successful in this regard, but now the general population is receiving more folic acid in the diet than ever before. Of concern is that excess folic acid intake will cause cancers to grow or metastasize more quickly than they would at lower levels of intake.

DNA undergoes a chemical modification, called methylation, which regulates the expression of genes. Folate metabolism produces compounds for this reaction. When folate levels are low, gene expression may change. Many cancers, including breast cancer, demonstrate gene expression differences compared to normal cells due to changes in methylation (i.e. hypermethylation). In addition, metastases show differences in gene expression compared to the primary breast tumor. If a cancer cell is dependent on a particular DNA methylation pattern to maintain its “cancerness” or metastatic potential, then manipulation of folic acid intake may impact the progression of cancer.

Our research project hypotheses are: 1) folate deficiency and the DNA-demethylating agent, 5-aza-deoxycytidine, will decrease incidence of metastasis; 2) excess dietary folate will increase incidence of metastasis; 3) folate deficiency and 5-aza-deoxycytidine will increase the expression of specific genes that are typically repressed in tumors; and 4) differences in gene expression in primary and metastatic tumor cells exposed to different levels of dietary folate and 5-aza-deoxycytidine will affect methylation status of specific genes. We will utilize a special mouse model (transplantable murine mammary intraepithelial neoplasia outgrowths or MIN-Os), which is a highly predictable system in terms of the time from transplantation to incidence of local tumor to incidence of metastasis. After breast tumor transplantation, the mice will be fed a diet that varies in folic acid compared to regular chow (the control diet). Some mice will receive weekly injections of 5-aza-deoxycytidine. We will use PET (positron emission tomography) scans to detect non-invasively tumor metastasis in the mice. Incidence of metastasis, microscopic tissue morphology (pathology), and analysis of gene expression differences on tumor samples will be compared among the treatment groups. We will compare tumor gene expression patterns in folate-deficient and 5-aza-deoxycytidine-treated mice with control and folate-excess mice. We will then investigate DNA sequences to determine gene-specific methylation status.

Currently, there are no effective treatments for breast cancer metastasis. The data from this project will potentially define how dietary intake of folic acid affects breast tumor metastasis.

Final Report (2010)

Note: the PI resigned the project after 2 years to pursue other career goals.

DNA undergoes a chemical modification, called methylation, which regulates the expression of genes. The methyl groups available for DNA methylation are manufactured by a B vitamin called folate which must be obtained from vegetables in the diet. When folate levels are low, changes in gene expression occur. In 1996, the US government mandated folate supplementation (in the form of folic acid) to cereals and grains to combat folate deficiency. The upper limits oftoxicity, however, were not established and folate supplementation levels in foods and "over the counter" vitamins were not regulated. The physiological consequences of excessive folate intake are unknown.

Metastatic breast cancer DNA has different methylation patterns compared to primary breast cancer. Changes in gene expression in primary mammary cancers may enable their metastatic potential and may be influenced by different levels of folate intake. Specific aim one compared the incidence and frequency ofmammary cancer metastasis using methods that change the methylation ofDNA in the primary cancer. We transplanted tumors into the mammary glands and fed the mice diets deficient, in excess and replete in folate for eleven weeks. These experiments tested the effects of excessive folate on metastasis using doses comparable to those available "over-the-counter" to the public population (800% of recommended daily values). We monitored the growth of metastasis in the lungs by micropositron emission tomography (microPET) imaging. We used a thymidine kinase probe that is detectable in proliferative tissues.

The imaging results after eleven weeks post-transplantation showed no lung metastasis in any of the mice. Specific aim two was designed to examine global DNA methylation in metastatic tumors and to compare gene expression by RNA microarray and methylation of specific genes. RNA microarrays detect which genes are "turned on and off" in response to the folate diets. Unfortunately, no metastases were detected in any of the control mice or experimental cohorts eleven weeks after transplantations. Hence, we were not able to retrieve any tissues for the RNA microarray.

In summary, the experiments in specific aim one were partially accomplished. Mice were fed diets having deficient and excessive levels of folate. However, no lung metastases were detected in any of the mice after eleven weeks post-transplantation of tumors. The second experiment extends the time for metastasis to twenty weeks post-transplantation of the tumors, the results of which are not yet known. The RNA microarray analysis was also not completed.