Epigenetic mechanisms of breast cancer prevention by the Type II diabetes mellitus agent metformin

Abstract

Breast cancer is the most commonly diagnosed cancer in the UK. Long-term use of metformin, a drug used to treat Type II Diabetes Mellitus, has been linked to a reduced risk of breast cancer in diabetic patients. This study aims to elucidate the mechanisms by which metformin acts as a potential breast cancer preventative agent in non-cancerous breast epithelial cells. Two models of normal breast epithelial cells were used; MCF12A and MCF10A non-cancerous breast epithelial cells lines, treated with 0mM, 2.5mM and 5mM metformin for 3-days in a range of metabolite levels (glucose, acetate, insulin and L-glutamine). Results were validated by primary normal matched breast epithelial and myoepithelial cells obtained from Breast Cancer Now Tissue Bank, treated with 1.5mM and 1mM metformin, respectively, for 7-days. RNA-sequencing and RT-qPCR, Illumina MethylationEPIC array and pyrosequencing, crystal violet, western blot and HILIC-MS were used to assess the mechanisms of metformin response in normal breast epithelial and myoepithelial cells. This study demonstrates sensitivity of normal breast epithelial cells to metformin (IC50 3mM-4mM), and low glucose (5mM) significantly modifies sensitivity to metformin (IC50 1mM-2mM). Gene expression is significantly altered in response to metformin, where 367 genes in MCF12A and 3 genes in MCF10A showed FDR<0.05. Overall, 7/8 selected gens were validated by RT-qPCR. Metformin subtly altered DNA methylation levels in a dose dependent manner. In addition, metformin appears to alter the expression of key methionine pathway genes. Metformin increases p-AMPK levels and decreases H3K27ac histone modification (0.5-fold, p=0.035). This is modified by glucose or acetate levels. However, effects of metformin appear to return to baseline following 24-hours. It is estimated that 23% of breast cancer cases could be prevented. This study highlights potential pathways metformin interacts with in normal breast epithelial cells.