P5-01-14: Phosphoproteomic Analysis of TIMP-1 Overexpressing MCF-7 Human Breast Cancer Cells Reveals Increased Expression and Phosphorylation of Topoisomerase Proteins.
Authors: Fogh L , Hekmat O , Munk S , Jensen NF , Brünner N , Stenvang J , Lademann U , Olsen JV 
Published: June 2012

Abstract
Background
Tissue inhibitor of metalloproteinase 1 (TIMP-1) is a protein with a potential biological role in drug resistance. Lack of TIMP-1 protein either alone (Willemoe et al., Eur J Cancer 2009) or in combination with Topoisomerase 2A gene aberrations has been shown to associate with increased benefit from adjuvant treatment with a Topoisomerase 2 inhibitor (epirubicin containing combination chemotherapy) while this association was not observed in patients treated with a non-Topoisomerase 2 inhibitor combination chemotherapy (Ejlertsen et al., JCO 2010).
Aim: To further investigate the molecular mechanisms underlying the association between TIMP-1 and epirubicin sensitivity by quantitative phosphoproteomics.
Methods: MCF-7 human breast cancer cells were transfected with pcDNA3.1(Hyg)-TIMP-1. Among 11 single cell clones, two TIMP-1 low expressing and two TIMP-1 high expressing clones were selected. The clones were labeled by SILAC (stable isotope labeling with amino acids in cell culture). Lysates were digested with trypsin and fractionated with SCX followed by subsequent enrichment of phosphopeptides by TiO2-based chromatography and desalting by C18 purification. Total peptides and phosphopeptides were analyzed by tandem mass spectrometry and quantified as described (JV Olsen et al., Science Signaling 2010). Selected proteins were confirmed on Western blots. The sensitivity of the four TIMP-1 cell clones was analyzed by treatment of the cells with the following drugs: The Topoisomerase 2 inhibitor epirubicin (an anthracycline). The Topoisomerase 1 inhibitor SN-38 (the active metabolite of irinotecan, a camptothecin analogue) and the combination of these. A specific Topoisomerase 2B inhibitor (XK 469, a quinoxaline analogue and the DNA crosslinker cisplatin. All experiments were determined with an endpoint MTT assay.
Results: The quantitative proteomic analyses confirmed the differences in TIMP-1 levels among the four clones. Several proteins were consistently found to be upregulated and/or had changed phosphorylation levels in the TIMP-1 high cells in two biological replicates. Of particular interest was the observation that the phosphorylation status and protein levels of Topoisomerase-1, -2A and -2B were increased in TIMP-1 high expressing cells compared to TIMP-1 low expressing cells. When the four clones were treated with specific Topoisomerase inhibitors, the TIMP-1 high expressing cells exhibited significantly more resistance to all three inhibitors compared to TIMP-1 low expressing cells. When cells were treated with a combination of SN-38 and epirubicin, we observed an additive but not a synergistic effect. At last, cells were treated with cisplatin with no different effect on TIMP-1 high and low expressing cells.
Conclusion and perspectives: The observed upregulation of both protein and phosphorylation levels of Topoisomerases in TIMP-1 high cells may be part of the mechanism by which TIMP-1 confers resistance to treatment with Topoisomerase inhibitors in primary breast cancer. Further work will include pathway analyses and hypothesis testing in clinical material.
Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-01-14.