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


METHODS
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 JNKpT183and BadpS112were 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.
NCT00930579.
Department of Medicine, College of Physicians and Surgeons, Columbia University Medical Center, New York, USA. kk2693@cumc.columbia.edu.|Herbert Irving Comprehensive Cancer Center, Columbia University, 161 Fort Washington Avenue, 10th Floor, Room 1069, New York, USA. kk2693@cumc.columbia.edu.
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http://orcid.org/0000-0002-1583-3553
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Kalinsky
Department of Statistics, Columbia University, New York, USA.
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T
Zheng
Herbert Irving Comprehensive Cancer Center, Columbia University, 161 Fort Washington Avenue, 10th Floor, Room 1069, New York, USA.|Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University Medical Center, New York, USA.
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Hibshoosh
Herbert Irving Comprehensive Cancer Center, Columbia University, 161 Fort Washington Avenue, 10th Floor, Room 1069, New York, USA.
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Du
Department of Medicine, College of Physicians and Surgeons, Columbia University Medical Center, New York, USA.
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P
Mundi
Department of Medicine, College of Physicians and Surgeons, Columbia University Medical Center, New York, USA.
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J
Yang
Department of Medicine, College of Physicians and Surgeons, Columbia University Medical Center, New York, USA.
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Refice
Herbert Irving Comprehensive Cancer Center, Columbia University, 161 Fort Washington Avenue, 10th Floor, Room 1069, New York, USA.|Department of Surgery, College of Physicians and Surgeons, Columbia University Medical Center, New York, USA.
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SM
Feldman
Herbert Irving Comprehensive Cancer Center, Columbia University, 161 Fort Washington Avenue, 10th Floor, Room 1069, New York, USA.|Department of Surgery, College of Physicians and Surgeons, Columbia University Medical Center, New York, USA.
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B
Taback
Herbert Irving Comprehensive Cancer Center, Columbia University, 161 Fort Washington Avenue, 10th Floor, Room 1069, New York, USA.|Department of Radiation Oncology, College of Physicians and Surgeons, Columbia University Medical Center, New York, USA.
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Connolly
Department of Medicine, College of Physicians and Surgeons, Columbia University Medical Center, New York, USA.|Herbert Irving Comprehensive Cancer Center, Columbia University, 161 Fort Washington Avenue, 10th Floor, Room 1069, New York, USA.|Department of Epidemiology and Biostatistics, Mailman School of Public Health, Columbia University, New York, USA.
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Crew
Department of Medicine, College of Physicians and Surgeons, Columbia University Medical Center, New York, USA.|Herbert Irving Comprehensive Cancer Center, Columbia University, 161 Fort Washington Avenue, 10th Floor, Room 1069, New York, USA.
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MA
Maurer
Department of Medicine, College of Physicians and Surgeons, Columbia University Medical Center, New York, USA.|Herbert Irving Comprehensive Cancer Center, Columbia University, 161 Fort Washington Avenue, 10th Floor, Room 1069, New York, USA.|Department of Epidemiology and Biostatistics, Mailman School of Public Health, Columbia University, New York, USA.
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Hershman
D017209:Apoptosis / Q000187:drug effects; D014408:Biomarkers, Tumor / Q000378:metabolism*; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D002285:Carcinoma, Intraductal, Noninfiltrating / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D002453:Cell Cycle / Q000187:drug effects; D015331:Cohort Studies; D005260:Female; D005500:Follow-Up Studies; D006801:Humans; D007004:Hypoglycemic Agents / Q000494:pharmacology; D007124:Immunoenzyme Techniques; D008687:Metformin / Q000494:pharmacology*; D009361:Neoplasm Invasiveness; D009367:Neoplasm Staging; D011379:Prognosis; D040081:Protein Array Analysis / Q000379:methods*; D040901:Proteomics / Q000379:methods*; D015398:Signal Transduction / Q000187:drug effects
D014408:Biomarkers, Tumor; D007004:Hypoglycemic Agents; D008687:Metformin
Breast Cancer; Cyclin D1; Immunohistochemistry; Metformin; Pre-surgical; Reverse Phase Protein Array