Effects of Metformin on Transcriptomic and Metabolomic Profiles in Breast Cancer Survivors Enrolled in the Randomized Placebo-Controlled MetBreCS Trial

Background: Metformin reduces the incidence of breast cancer in patients with type 2 diabetes and obesity. However, our knowledge about the effects of metformin on cancer recurrence in breast tissue is limited. Therefore, in this study, we examined the breast tissue gene expression changes by metformin in breast cancer survivors. Methods: Within the randomized placebo-controlled MetBreCS trial, baseline and one-year post-treatment fasting plasma and serum as well as breast tissue biopsies were collected. Breast cancer survivors with BMI > 25 kg/m 2 were randomly assigned to metformin (n=27), or placebo (n=13). We analyzed the transcriptomic profiles of the tissue biopsies by RNA sequencing. We also performed high-throughput metabolomics and sex steroid hormone analyses on the plasma and serum samples, respectively. To identify the metformin-associated signaling pathways in breast tissues, we integrated the gene expression and metabolomics and steroid hormone profiles using bivariate and functional analysis. Results: Comparing breast tissue transcriptomic data, we identified MS4A1 , HBA2 , MT-RNR1 and MT-RNR2 expression to be differentially expressed in breast tissues from pre- and postmenopausal women. We also found significant metformin-associated down-regulation of EGFL6 and FDCSP in postmenopausal women. Long-term metformin treatment was significantly associated with decreased plasma levels of citrulline, arginine, PC ae C36:5, PC ae C38:6, caffeine, and 4-methyl-2-oxovalerate. The integration of transcriptomic and metabolomic profiles using bivariate correlation analysis followed by functional analysis revealed a down-regulation of immune response associated with the reduced plasma levels of arginine and citrulline in the pre- and postmenopausal metformin-treated group. The correlation between two steroid hormones (17β-estradiol, estrone) and global gene expression also showed an enrichment of steroid hormone biosynthesis and metabolism pathway with highly negatively correlated CYP11A1 and CYP1B1 expression in breast tissue from postmenopausal metformin-treated women. Conclusions: Our results indicate that breast cancer survivors treated with metformin have specific changes in breast tissue gene expression that may prevent the development of new tumors. Reduced levels of circulating arginine, citrulline, and estrogens in metformin-treated breast cancer survivors may also contribute to reducing recurrence risk in obesity-associated breast cancer. Trial registration MetBreCs trial was started in 2015 and is registered at European Union Clinical Trials Register (EudraCT Protocol #: 2015-001001-14) on 7 October 2015.