ABSTRACT
BACKGROUND: Studies in taxane and/or anthracycline refractory metastatic breast cancer (mBC) patients have shown approximately 30% response rates to irinotecan. Hence, a significant number of patients will experience irinotecan-induced side effects without obtaining any benefit. The aim of this study was to lay the groundwork for development of predictive biomarkers for irinotecan treatment in BC. METHODS: We established BC cell lines with acquired or de novo resistance to SN-38, by exposing the human BC cell lines MCF-7 and MDA-MB-231 to either stepwise increasing concentrations over 6 months or an initial high dose of SN-38 (the active metabolite of irinotecan), respectively. The resistant cell lines were analyzed for cross-resistance to other anti-cancer drugs, global gene expression, growth rates, TOP1 and TOP2A gene copy numbers and protein expression, and inhibition of the breast cancer resistance protein (ABCG2/BCRP) drug efflux pump. RESULTS: We found that the resistant cell lines showed 7-100 fold increased resistance to SN-38 but remained sensitive to docetaxel and the non-camptothecin Top1 inhibitor LMP400. The resistant cell lines were characterized by Top1 down-regulation, changed isoelectric points of Top1 and reduced growth rates. The gene and protein expression of ABCG2/BCRP was up-regulated in the resistant sub-lines and functional assays revealed BCRP as a key mediator of SN-38 resistance. CONCLUSIONS: Based on our preclinical results, we suggest analyzing the predictive value of the BCRP in breast cancer patients scheduled for irinotecan treatment. Moreover, LMP400 should be tested in a clinical setting in breast cancer patients with resistance to irinotecan.
Subject(s)
ATP-Binding Cassette Transporters/genetics , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Camptothecin/analogs & derivatives , DNA Topoisomerases, Type I/genetics , Neoplasm Proteins/genetics , ATP Binding Cassette Transporter, Subfamily G, Member 2 , ATP-Binding Cassette Transporters/biosynthesis , Antigens, Neoplasm/genetics , Breast Neoplasms/pathology , Camptothecin/administration & dosage , Camptothecin/adverse effects , DNA Topoisomerases, Type I/biosynthesis , DNA Topoisomerases, Type II/genetics , DNA-Binding Proteins/genetics , Docetaxel , Drug Resistance, Neoplasm/genetics , Female , Gene Dosage/genetics , Gene Expression Regulation, Neoplastic/drug effects , Humans , Irinotecan , MCF-7 Cells , Neoplasm Proteins/biosynthesis , Poly-ADP-Ribose Binding Proteins , Taxoids/administration & dosageABSTRACT
Tissue inhibitor of metalloproteinase 1 (TIMP-1) is a protein with a potential biological role in drug resistance. To elucidate the unknown molecular mechanisms underlying the association between high TIMP-1 levels and increased chemotherapy resistance, we employed SILAC-based quantitative mass spectrometry to analyze global proteome and phosphoproteome differences of MCF-7 breast cancer cells expressing high or low levels of TIMP-1. In TIMP-1 high expressing cells, 312 proteins and 452 phosphorylation sites were up-regulated. Among these were the cancer drug targets topoisomerase 1, 2A, and 2B, which may explain the resistance phenotype to topoisomerase inhibitors that was observed in cells with high TIMP-1 levels. Pathway analysis showed an enrichment of proteins from functional categories such as apoptosis, cell cycle, DNA repair, transcription factors, drug targets and proteins associated with drug resistance or sensitivity, and drug transportation. The NetworKIN algorithm predicted the protein kinases CK2a, CDK1, PLK1, and ATM as likely candidates involved in the hyperphosphorylation of the topoisomerases. Up-regulation of protein and/or phosphorylation levels of topoisomerases in TIMP-1 high expressing cells may be part of the mechanisms by which TIMP-1 confers resistance to treatment with the widely used topoisomerase inhibitors in breast and colorectal cancer.
Subject(s)
Drug Resistance, Neoplasm , Protein Processing, Post-Translational , Proteome/metabolism , Tissue Inhibitor of Metalloproteinase-1/physiology , Amino Acid Sequence , Antineoplastic Agents/pharmacology , Breast Neoplasms , Cell Survival/drug effects , Cisplatin/pharmacology , Consensus Sequence , DNA Topoisomerases, Type I/chemistry , DNA Topoisomerases, Type I/metabolism , DNA Topoisomerases, Type II/chemistry , DNA Topoisomerases, Type II/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Female , Gene Expression , Humans , MCF-7 Cells , Molecular Sequence Data , Phosphorylation , Protein Interaction Maps , Proteome/chemistry , Topoisomerase Inhibitors/pharmacologyABSTRACT
We and others have shown central roles of the Na(+)/H(+) exchanger NHE1 in cell motility. The aim of this study was to determine the roles of NHE1 and of the Na(+), HCO(3)(-) cotransporter NBCn1 in motility of serum-starved MCF-7 breast cancer cells expressing constitutively active ErbB2 (ΔNErbB2). ΔNErbB2 expression elicited NBCn1 upregulation, Ser(703)-phosphorylation of NHE1, and NHE1-inhibitor (EIPA)-sensitive pericellular acidification, in conjunction with increased expression of ß1 integrin and ERM proteins. Active ERM proteins and NHE1 colocalized strongly to invadopodial rosettes, the diameter of which was increased by ΔNErbB2. Adhesion and migration on collagen-I were augmented by ΔNErbB2, unaffected by the NBC inhibitor S0859, and further stimulated by EIPA in a manner potentiated by PI3K-Akt-inhibition. These findings demonstrate that NHE1 inhibition can enhance cancer cell motility, adding an important facet to the understanding of NHE1 in cancer.
