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

Abstract

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 pMAPKpT202,Y204, JNKpT183,pT185, BadpS112, PKC.alphapS657, and SrcpY416. Cyclin D1 change after metformin by IHC was not observed. In cell lines, reductions in JNKpT183 and BadpS112 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.

Keywords: Breast Cancer; Cyclin D1; Immunohistochemistry; Metformin; Pre-surgical; Reverse Phase Protein Array.

Conflict of interest statement

Conflict of interest There are no conflicts of interest to declare for this study.

Figures

Fig. 1

Median-centered unsupervised hierarchical cluster of breast cancer samples (pre-/post-treatment paired samples from 32 metformin-treated cases and 34 untreated matched historical controls) using quantification data for 161 proteins derived from reverse phase protein arrays (RPPA)

Fig. 2

Changes in protein expression by the western blot analysis after treatment with metformin (up to 4 mM) There was a dose-dependent decrease in JNKpT183 and BadpS112in both 2a) T47D and 2b) MDA-MB-468 cells. An increase in AMPKpT72 was observed. No change in raptor or cyclin D1 was identified. Actin was a control. Met metformin