Proteomic modulation in breast tumors after metformin exposure: results from a “window of opportunity” trial

Purpose: Reverse Phase Protein Array (RPPA) is a high-throughput antibody-based technique to assess cellular protein activity. The goal of this study was to assess protein marker changes by RPPA in tumor tissue from a pre-surgical metformin trial in women with operable breast cancer (BC). Methods: In an open-label trial, metformin 1500-mg PO daily was administered prior to resection in 35 non-diabetic patients with stage 0–III BC, body mass index ≥25 kg/m². For RPPA, formalin-fixed paraffin-embedded (FFPE) samples were probed with 160 antibodies. Paired and two-sample t-tests were performed (p ≤ 0.05). Multiple comparisons were adjusted for by fixing the false discovery rate at 25 %. We evaluated whether pre- and post-metformin changes of select markers by RPPA were identified by immunohistochemistry (IHC) in these samples. We also assessed for these changes by western blot in metformin-treated BC cell lines. Results: After adjusting for multiple comparisons in the 32 tumors from metformin-treated patients vs. 34 untreated historical controls, 11 proteins were significantly different between cases vs. controls: increases in Raptor, C-Raf, Cyclin B1, Cyclin D1, TRFC, and Syk; and reductions in pMAPK^pT202,Y204, JNK^pT183,pT185, Bad^pS112, PKC.alpha^pS657, and Src^pY416. Cyclin D1 change after metformin by IHC was not observed. In cell lines, reductions in JNK^pT183 and Bad^pS112 were seen, with no change in Cyclin D1 or Raptor. Conclusions: These results suggest that metformin modulates apoptosis/cell cycle, cell signaling, and invasion/motility. These findings should be assessed in larger metformin trials. If confirmed, associations between these changes and BC clinical outcome should be evaluated. Clinicaltrials.gov identifier: NCT00930579.