Abstract B79: Multidrug resistance protein 1 (MRP-1) is expressed in the mitochondria of normal and cancer cells and may play a role in intrinsic drug resistance of ESFT cells.

Abstract

Abstract Overexpression of plasma membrane MRP-1 can lead to multidrug resistance. In this study we describe for the first time the expression of mitochondrial MRP-1 in untreated human normal and cancer cells and tissues and investigate the functional significance of mitochondrial MRP-1 with a view to identifying a new therapeutic starategy. MRP-1 expression and subcellular localisation in normal and cancer cells and tissues was examined by differential centrifugation and western blotting, and immunofluorescence microscopy. Protein extracts were de-glycosylated using the Protein Deglycosylation Mix (NEB). Viable mitochondria were isolated and MRP-1 efflux activity measured using the calcein-AM functional assay. MRP-1 expression was increased by retroviral infection and specific overexpression confirmed by RNA array. Cell viability was determined by trypan blue exclusion and annexin V-PI labelling of cells. Mitochondrial membranes were solubilised by incubation with increasing concentrations of the detergent digitonin and MRP-1 localisation within the mitochondria evaluated by Western blot; protein markers of the mitochondrial outer and inner membranes, the matrix and intermembrane space were used to confirm localisation within the mitochondria. For the first time we have detected MRP-1 in the mitochondria of untreated cancer and normal cells and tissues by Western blot and confirmed these findings by immunofluorescence and confocal microscopy. The size of the MRP-1 protein in the mitochondria of cancer and normal cells was between 150–250 kDa, reflecting differences in glycosylation. The efflux activity of mitochondrial MRP-1 was more efficient (55–64%) than plasma membrane MRP-1 (11–22%; p<0.001). Specific overexpression of MRP-1 in TC-32 cells by retroviral infection induced a preferential increase in mitochondrial MRP-1 (150%), resulting in an 85% increase in MRP-1 efflux activity (p<0.0001), suggesting selective targeting to this organelle. This was associated with an increase in the IC50 of the MRP-1 substrates vincristine (4.5nM to 14.7nM; p<0.001) and etoposide (101.7nM to 121.6nM (p<0.001) but not the non MRP-1 substrate actinomycin D (0.5nM). Treatment with a non-lethal concentration of doxorubicin (0.85nM, 8h) increased mitochondrial and plasma membrane MRP-1, increasing resistance to MRP-1 substrates. The glycosylation of MRP-1 and the efflux of calcein-F from whole mitochondria are consistent with localisation of MRP-1 to the outer mitochondrial membrane. This was confirmed by the increased expression of MRP-1 protein in the mitochondrial supernatant after treatment with digitonin and centrifugation. The expression pattern of MRP-1 was similar to that of porin, an outer mitochondrial membrane marker. Analysis of the MRP-1 sequence (using Predator and MitoProtII) failed to reveal published mitochondrial targeting sequences, and so we are currently investigating the mechanism of MRP-1 transport to the mitochondria to identify a druggable therapeutic target. MRP-1 expressed in the outer mitochondrial membrane has increased efflux activity compared to plasma membrane MRP-1, and so represents an exciting new therapeutic target where historically conventional MRP-1 inhibitor strategies targeting plasma membrane MRP-1 have had limited clinical success. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B79.