Abstract P4-15-03: Proteomic modulation in breast tumors after metformin use: Results from a “window of opportunity” trial
Authors: Kalinsky K , Zheng T , Crew KD , Refice S , Feldman SM , Taback B , Hibshoosh H , Su T , Maurer MM , Hershman DL 
Published: August 2014

Abstract
Background: Laboratory and population studies demonstrate that metformin offers a beneficial breast cancer (BC) effect through reduction of serum insulin levels and changes in cellular protein synthesis and growth, such as AMPK pathway signaling. In a pre-surgical metformin trial of overweight/obese, multi-ethnic BC patients, we reported no difference in tumor proliferation, as measured by ki-67. However, reductions in other biomarkers were observed, including reduction in body mass index (BMI), serum cholesterol, serum insulin, and leptin. Reverse Phase Protein Array (RPPA) is a high-throughput antibody-based technique to assess cellular protein activity in signaling networks. The goal of this study was to assess changes in functional proteomics through RPPA in patients treated in a pre-surgical metformin trial.
Methods: Metformin 1500mg PO daily (500mg am/1000 mg pm) was administered for 2-4 weeks prior to resection in 35 patients with stage 0-III operable BC, BMI ≥ 25 kg/m2, and no history of diabetes. Protein was extracted from pre- and post-metformin paraffin-embedded tumor tissue, denatured by sodium dodecyl sulfate, and printed on nitrocellulose-coated slides. Samples were probed with 160 antibodies. Evaluated antibodies associated with various cellular activities, including PI3K/AKT signaling, HSP90 clients, Src/STAT activity, and apoptosis. We analyzed changes in RPPA parameters in tumor tissue of study patients with those of untreated historical controls, matched by age, BMI, and tumor characteristics. Paired t-test was used to calculate within-group changes in RPPA, and two-sample t-tests were used to compare between-group changes in cases and controls (significance: p ≤ 0.05). Multiple comparisons were adjusted for by fixing the false discovery rate (FDR) at 25%.
Results: Of the 35 metformin-treated patients, 32 were evaluable. The majority were Hispanic (80%). Metformin was administered for a median of 23 days (range: 8-64). Of the invasive BCs (n = 21/35), 80% of patients had HR+/HER2- BC. The 33 historical controls were well-matched. For RPPA, the mean total formalin-fixed paraffin embedded protein concentration was 38.9 ug (SD: 3.3). Of the 160 antibodies, 67 antibodies significantly changed after metformin use in the treated group, including reduction in pAKTS473, pAKTT308, and MTOR (unadjusted). Nineteen antibodies were identified as having between group differences in change from baseline: increase in BadpS112, C-RAF, Claudin-7, Cyclin B1, Cyclin D1, EGFR, HER3 pY1298, Lck, PKC-alphapS657, RAD50, Raptor, Syk, and TRFC; reduction in 14-3-3 epsilon, FOXO3a, JNKpT183, MAPKpT202, MEK1pS217, and SrcpY416. Adjusting for multiple comparisons, the following remain statistically significantly different between cases vs. controls: increase in BadpS112, C-RAF, Cyclin D1, and Raptor; and reduction in JNKpT183. Further assessment of RPPA modulation is ongoing, including changes grouped by signaling pathway and activity, as well as validation by immunohistochemistry.
Conclusions: Compared to controls, metformin use associates with changes in apoptosis, cell signaling, C-RAF, and Raptor. These results should be further validated in larger metformin intervention trials to better define populations who may benefit from this therapy.
Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-15-03.