The Activated MEK/ERK Pathway may Potentiate Breast Cancer Resistance Protein /ABCG2 Function in SN38-selected MCF-7 Cells

A variant of the breast cancer MCF-7 cells, termed MCF-7/SNR, was established by chronic and step-wise exposure to SN38, an active metabolite of irinotecan. MCF7/SNR cells were >100-fold more resistant to SN38 than parental MCF-7 cells, while resistances to vincrisitine and cisplatin were similar between those cells. MCF-7/SNR cells overexpressed endogenous breast cancer resistance protein (BCRP)/ABCG2 that efflux SN38 and topotecan out of cells, but the expression levels were similar to those of exogenous BCRP-transfected MCF-7/BCRP cells which were approximately 18-fold more resistant than the parental cells. In addition, Ko-143, a specific BCRP inhibitor, completely reversed the resistance, suggesting that the high degree of SN38 resistance of MCF-7/SNR cells was attributable to overexpressed BCRP for the most part. cDNA microarray and reverse transcription-PCR analyses revealed that the RASGRP1 and jak-2 genes, both associated with activation of the Ras/Raf/MEK/ ERK pathway, were markedly up-regulated in MCF-7/SNR cells. Consistently, seruminduced phosphorylation of ERK1/2 was found to be enhanced in MCF-7/SNR cells than in MCF-7 cells. Furthermore, MEK inhibitors, PD98059 and U0126, partially reversed SN38 resistance of MCF-7/SNR cells. Treatment with PD98059 scarcely affected BCRP expression levels but mildly increased cellular topotecan uptake in MCF-7/SNR cells. Finally, cytoplasmic BCRP distribution was found to be increased in MCF-7/SNR cells after PD98059 treatment. These data suggest that activation of the MEK/ERK pathway may potentiate BCRP function by regulating its trafficking and turnover. Central Imai et al. (2014) Email: JSM Clin Oncol Res 2(1): 1008 (2014) 2/7 result in apoptosis. Irinotecan is used in the treatment of certain neoplasm such as inoperable and/or recurrent colorectal cancers, esophageal cancers, gastric cancers, non-small cell and smallcell lung cancers, non-Hodgkin lymphoma, and breast cancers [1-3]. In spite of an initial response, most patients treated with irinotecan become resistant to this chemotherapy. Therefore, we aimed to determine the mechanism involved in resistance to SN38 in human cancer cells. To this end, we exposed human breast cancer MCF-7 cells to SN38 continuously and progressively and obtained cells 160-fold more resistant than the parental cells (MCF-7/SNR). Breast cancer resistance protein (BCRP)/ABCG2 transports SN38 across the cell membrane out of cells [4]. Although MCF7/SNR cells endogenously expressed as much BCRP as MCF-7/ BCRP cells that overexpressed exogenous BCRP, MCF-7/SNR cells were far more resistant than MCF-7/BCRP cells. The higher levels of SN38 resistance of MCF-7/SNR cells were attributed to endogenous BCRP, since Ko-143, a specific BCRP inhibitor, completely reversed the resistance [5]. Further analyses revealed that the Ras/Raf/mitogen activated-protein kinase/ERK kinase (MEK)/ extracellular signal-regulated kinase (ERK) pathway was activated in MCF-7/SNR cells and that MEK inhibitors partially circumvented the SN38 resistance. Treatment of MCF-7/SNR cells with PD98059 resulted in mild increase of intracellular accumulation of topotecan, which is another substrate of BCRP, without affecting BCRP levels [6]. In addition, alteration of cellular BCRP distribution was observed after PD98059 treatment. These data suggest that activation of the MEK/ERK pathway may regulate BCRP trafficking and turnover, and potentiate BCRP function on the plasma membrane. MATERIALS AND METHODS