Purpose: Over-expression of multi-drug resistance protein 1 (MRP1) predicts poor outcome for children with neuroblastoma (NB) (Haber et al, 2006, JCO, 24, 1546-53). MRP1 is expressed in a number of different subcellular organelles and is a reported client protein of the heat shock protein (HSP) family (Roundhill et al, 2016, FASEB, Epub, PMID:26722004). We have investigated the expression, transport and activity of MRP1 in NB.
Methods: MRP1 expression was examined by subcellular fractionation and western blotting (WB), electron microscopy (EM) and immunofluorescent (IF) microscopy. Expression was increased following infection with an MRP1 expressing retrovirus. MRP1 efflux activity was evaluated using calcein-AM and disrupted using valinomycin. Viable cell number was measured by trypan blue exclusion and cell migration examined in a wound healing and 3D assay. Interactions between MRP1 and HSPs were investigated by immunoprecipitation, and inhibition of HSP90 using NVPAUY and 17-AAG.
Results: MRP1 protein and efflux activity were detected in NB cell lines (12/12). High MRP1 expression was associated with MYCN amplification (4/6). MRP1 was detected in the plasma membrane, nucleus and mitochondria of NB cells and tumours. Disruption of mitochondrial MRP1 activity did not enhance vincristine (MRP1 substrate) cytotoxicity in the MYCN and MYCC amplified cells (CI >0.9), although it did increase vincristine-induced cell death in the non-amplified cells (CI <0.7). Resistance to MRP1 substrates (p>0.05), cell doubling time (p>0.05) and migration (p>0.05) were unchanged after increased expression of MRP1.
NB cells expressed the chaperone proteins HSP70, HSP90α and HSP90β. MRP1 co-precipitated with HSP90β. NVPAUY (20-1280nM) significantly decreased viable cell number by 44-59% (>160nM; p<0.05) in NB cells, whereas 17-AAG (30-200nM), a less potent HSP90 inhibitor, had no effect on NB cell number (p>0.05 at all concentrations examined).
Conclusions: MRP1 is expressed in the mitochondria of NB cells and may be a client protein for HSP90β. The clinical significance of mitochondrial MRP1 in NB requires further investigation.