Abstract 1921: Low doses of metformin increase expression of microRNAs that regulate innate immune genes in breast cancer cells
Authors: Woodard Jonne S. , Yeyeodu Susan , Kimbro Kevin S. 
Published: July 2016

Background: Metformin (MF), the most widely used anti-diabetic drug, has also been shown to impact the incidence of breast cancer (BrCa), although the molecular mechanisms responsible for the therapeutic effects of MF are poorly understood. Unfortunately, until now in vitro studies have used high doses of MF (5-20 mM) and may not accurately model MF mechanisms of action at clinical doses.

Objectives & Hypothesis: Metformin is thought to inhibit cancer cell growth by decreasing available cellular energy, creating a fatal metabolic deficit in cancer cells. Recent evidence suggests that innate immunity may play a role in the regulation of metabolism. We hypothesize that: 1) metabolic pathways targeted by MF impact innate immune pathways modulated by IRAK4 in BrCa cells and 2) metabolic and innate immune pathways in BrCa are regulated by common microRNAs (miRs). Therefore, we examined the effects of a) pharmacologically relevant MF concentrations on the expression of selected miRs and proteins associated with innate immunity and metabolism, and b) various concentrations of MF on BrCa cell lines that differ in their metastatic potential.

Methods: Highly metastatic 231s and moderately metastatic 468s were cultured in low glucose medium and 15uM MF for 0, 2, 6, 12 and 24hrs. Changes in the expression of 43 miRs associated with innate immune and/or metabolic pathways were determined from total RNA by qRT-PCR using a miR array. MF-induced changes in IRAK4 protein expression were compared with known MF targets AMPK and its upstream regulator LKB1. The effects of MF concentrations up to 1 mM on BrCa cell growth and migration were also tested.

Results: MF enhanced levels of hsa-miR-124-3p (which targets LKB1and AMPK) &gt;2-fold at 2 and 6hrs in 468s. In contrast, hsa-miR-302c (which targets IRAK4) was up-regulated (&gt;2-fold) by MF in 231s but not 468s at 2 and 6hrs. Expression of total IRAK4 protein was reduced after 12hrs of exposure to MF in 231s and after 6 and 12 hours in 468s. As reported previously, LKB1 was not expressed in 231s, whereas LKB1 was constitutively expressed in 468s. AMPK activation was inhibited at 2 and 12hrs in 231s, while in 468s AMPK activation was highly variable. Neither cell line showed changes in proliferation or migration in the presence of MF.

Conclusions: Low concentrations of MF were sufficient to increase levels of two miRs known to regulate innate immune transcripts. MF treatment reduced IRAK4 levels by 6hrs (468s) to 12hrs (231s). Cell-specific differences in AMPK and LKB1 activation likely reflect differences in their metabolic rates and programs. Our data suggests that MF-sensitive metabolic pathways are involved in IRAK4-dependent innate immunity in breast cancer cells. Future studies will continue to unravel the effects of low doses of MF on BrCa.

Citation Format: Jonne S. Woodard, Susan Yeyeodu, Kevin S. Kimbro. Low doses of metformin increase expression of microRNAs that regulate innate immune genes in breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1921.