Molecular cytogenetic characterization of doxorubicin‐resistant neuroblastoma cell lines: Evidence that acquired multidrug resistance results from a unique large amplification of the 7q21 region

Abstract

Neuroblastoma is a heterogeneous neural crest-derived embryonic childhood neoplasm that is the second most common solid tumor found in children. Despite recent advances in combined therapy, the overall survival of patients with high-stage disease has not improved in the last decades. Treatment failure is in part attributed to multidrug resistance. To address the mechanisms involved in the development of multidrug resistance, we have generated two doxorubicin-resistant neuroblastoma cell lines (IGRN-91R and LAN-1R). These cells were shown to overexpress the MDR1 gene coding for the P-glycoprotein and were resistant to other MDR1- and non-MDR1-substrate drugs. Indeed, the MDR1 inhibitor verapamil only partially restored sensitivity to drugs, confirming that P-glycoprotein-mediated drug efflux was not responsible for 100% resistance. High-resolution and array-based comparative genomic hybridization analyses revealed the presence of an amplicon in the 7q21 region as the unique genomic alteration common to both doxorubicin-resistant cell lines. In addition to the MDR1 locus, this large amplified region is likely to harbor additional genes potentially involved in the development of drug resistance. This study represents the first molecular cytogenetic and genomic approach to identifying genomic regions involved in the multidrug-resistant phenotype of neuroblastoma. These results could lead to the identification of relevant target genes for the development of new therapeutic modalities.

The Wnt receptor FZD1 mediates chemoresistance in neuroblastoma through activation of the Wnt/β-catenin pathway

“…We have recently reported that DoxR NB cell lines exhibited overexpression of the MDR1 gene due to an amplification at the 7q21 locus. As the MDR1/P-gp inhibitor verapamil was not able to restore 100% of cell sensitivity to DoxR, etoposide or paclitaxel, we postulated that P-glycoprotein-mediated drug efflux was not responsible for 100% drug resistance (Flahaut et al, 2006b). In this study, microarray expression profile analysis of resistant variants revealed 7q21 region-related amplification and overexpression of several genes, including MDR1 and the Wnt receptor FZD1.…”

“…Interestingly, FZD1 encoding the frizzled 1 receptor, a seven transmembrane receptor member of the Wnt/b-catenin signalling pathway was found to be the highest upregulated transcript (34.5-fold stimulation) in resistant cells. Moreover, 12 out of 16 transcripts in the list of upregulated transcripts corresponded to genes located on the 7q21 region as published earlier (Flahaut et al, 2006b). As the 7q21 amplified region also harbours the FZD1 gene, we now investigated whether the two MDR1 and FZD1 genes were coamplified.…”

“…In NB as well, high levels of MDR1 mRNA are often associated with acquired resistance to chemotherapy (Goldstein et al, 1990) and thus poor survival of the patients. We recently reported that two chemoresistant NB cell lines, generated by prolonged exposure to doxorubicin, acquired a similar amplification on chromosome 7 (7q21 amplicon), harbouring the region of the MDR1 gene (Flahaut et al, 2006b). A strong increase of MDR1 expression was in consequence measured in both resistant variants.…”

“…A strong increase of MDR1 expression was in consequence measured in both resistant variants. However, acquired chemoresistance is not restricted to MDR1/P-gp overexpression as sensitivity could only partially be restored by the addition of the P-gp inhibitor verapamil, and resistance to cisplatin is not mediated by P-gp-mediated efflux (Flahaut et al, 2006b).…”

“…To elucidate genes and pathways involved in chemoresistance in NB cells, we have further analysed chemoresistant NB cell lines generated by prolonged exposure to doxorubicin (DoxR cells; Flahaut et al, 2006b). We have recently reported that DoxR NB cell lines exhibited overexpression of the MDR1 gene due to an amplification at the 7q21 locus.…”