Abstract PD03-05: Analysis of tumour cell signaling in response to neoadjuvant metformin in women with early stage breast cancer.
Authors: Dowling RJO , Niraula S , Chang MC , Done SJ , Ennis M , Hood N , McCready DR , Leong W , Escallon JM , Reedijk M , Goodwin PJ , Stambolic V 
Published: August 2014

Abstract
Background: The anti-diabetic drug metformin, commonly used to treat type 2 diabetes due to its ability to reduce circulating glucose and insulin, has emerged as a potential anti-cancer agent. Observational studies have reported decreased cancer incidence and mortality in diabetics receiving metformin. Metformin's ability to reduce insulin may be particularly important for breast cancer (BC) because hyperinsulinemia is an adverse prognostic factor and most cells express the insulin receptor (IR). The anti-cancer effects of metformin are associated with both direct (insulin-independent) and indirect (insulin-dependent) actions. Direct effects are linked to activation of AMPK and an inhibition of mTOR signalling, while indirect effects are mediated by reductions in circulating insulin levels, leading to reduced IR-activated PI3K signalling. We conducted a neoadjuvant, single arm, “window of opportunity” trial examining the clinical and biological effects of metformin on thirty-nine locoregional BC patients awaiting definitive surgery.
Methods: Non-diabetic women with newly diagnosed, untreated BC were given metformin 500 mg tid for ≥2 weeks post diagnostic core biopsy until surgery. Fasting blood and tumour samples were collected at diagnosis and surgery. Blood glucose and insulin were assayed to assess the physiologic effects of metformin, while IHC analysis of tumours was used to characterize cellular markers before and after metformin. Specifically, IR levels and the phosphorylation status of proteins involved in AMPK and PI3K/AKT/mTOR signalling, including AMPK (T172) and AKT (S473), were examined.
Results: 39 patients with a mean age of 51 years received metformin for a median of 18 days (range 13–40) with minor GI toxicities. The clinical effects (previously reported) included significant (p &lt; 0.05) decreases in body mass index (−0.5 kg/m2), weight (−1.2 kg), glucose (−0.14 mM) and HOMA (an estimate of insulin resistance, −0.21), and a decrease in insulin (−4.7 pmol/L) that approached significance (p = 0.0686). Ki67 staining in tumour tissue decreased significantly and TUNEL increased significantly. Levels of IR expression decreased significantly (from 4.39 to 3.82, p = 0.0375) as did the phosphorylation status of AKT (S473) and AMPK (T172) (from 9.82 to 7.08, p = &lt;0.0001; from 6.2 to 5.1, p = 0.0034, respectively).
Conclusions: Metformin impact was consistent with beneficial anti-cancer effects. Reduced AKT phosphorylation, coupled with decreased insulin and IR levels, suggest insulin-dependent effects are important in the clinical setting. Assessment of additional factors in BC cells, including OCT1 expression (required for metformin uptake), and the phosphorylation of ACC (a marker of AMPK activation), is underway and will be reported. Integrated analysis of these factors combined with the physiological and molecular data described above will further enhance understanding of metformin action in the clinical setting.
Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr PD03-05.