MYCN enhances P-gp/MDR1 gene expression in the human metastatic neuroblastoma IGR-N-91 model

Abstract

Despite intensive high-dose chemotherapy and autologous hematopoietic stem cell transplantation, disseminated neuroblastoma (NB) frequently proves to be chemosensitive but not chemocurable, and more often so in NB-presenting MYCN amplification. To assess the direct relationship between the MYCN oncogene and chemoresistance acquisition during NB metastatic dissemination, we have studied MYCN and MDR1 genes using the human IGR-N-91 ectopic xenograft metastatic model. This characterized experimental in vitro model includes human neuroblasts derived from a subcutaneous primary tumor xenograft, disseminated blood cells, myocardium, and bone marrow (BM) metastatic cells. All IGR-N-91-derived neuroblasts harbor a consistent MYCN genomic content but, unlike primary tumor xenograft, BM, and myocardium, human neuroblasts elicit a concomitant increase in MYCN and MDR1 transcripts levels, consistent with chemoresistance phenotype and active P-gp. In contrast, no variation of MRP1 transcript level was associated with the metastatic process in this model. Using an MDR1 promoter-CAT construct, we have shown that the MycN protein activates MDR1 transcription both in exogenous transient MYCN-transfected SK-N-SH cells and in endogenous BM metastatic neuroblasts with an increase in the MYCN transcript level. Band-shift experiments indicate that IGR-N-91 cells enriched with the MycN transcription factor do bind to two E-box motifs localized within the MDR1 promoter. Overall, our data indicate that MYCN overexpression increment contributes to the acquired drug resistance that occurs throughout the NB metastatic process.

Figure 1.

The IGR-N-91-derived human xenograft NB model. The IGR-N-91 cell line was established from an involved BM collected from a high-risk NB (stage 4-NB, 8-year-old boy). Neuroblasts were injected subcutaneously into nude mice and a PTX was isolated. The PTX neuroblasts were then minced to be subcutaneously xenografted to other mice. This procedure was repeated three times. Blood-disseminated as well as metastatic neuroblasts from the Myoc and BM were then cultured on bovine corneal extracellular matrix. These malignant human neuroblasts were further subcultured in vitro to established cell lines: PTX and blood cells, Myoc, and BM metastatic sublines.

Figure 2.

Analysis of MYCN and multi-drug resistance gene expression by RTQ-PCR (A) and Northern blotting (B) in NB cell lines. MYCN (1/100 mRNA expression/18S), MDR1 and MRP1 mRNA levels are shown. The increase in the MYCN gene mRNA level is correlated with the increase in the MDR1 gene mRNA level in the IGR-N-91 xenograft model for Myoc and BM metastatic neuroblasts. No variations in MRP1 gene expression in cell lines is determined by RTQ-PCR or Northern blotting. Data are the mean ± SD of three independent experiments.

Figure 3.

A: Western blots of MycN, c-myc, P-gp, P53, and Bcl2 proteins in NB cell lines. MycN protein expressions are well correlated with mRNA gene expressions in NB cell lines. A higher level of MycN protein expression is noted in BM and Myoc neuroblasts. Note the absence of c-myc protein expression in MYCN-overexpressing neuroblasts and of MycN protein in c-myc expressing neuroblasts. Wild-type p53 is present in SK-N-SH neuroblasts in contrast to IGR-N-91, PTX, and metastatic sublines, which all present similar heavier shifted-p53 protein (arrow). B: Flow cytometric analysis of Rho-123 uptake in NB cell lines. P-gp expression is determined by the uptake/efflux ratio of rhodamine-123. The cutoff value of this ratio was arbitrarily fixed at 2. A representative histogram of mean fluorescence intensity is shown for the different cell lines. Controls are KB3–1 and KB-A1 cell lines.

Figure 4.

Response of NB cell lines to VP16 or CDDP cytotoxicity. A: Viability of NB cells treated throughout a concentration range of 0 to 30 μmol/L VP16 or CDDP for 48 hours was determined by MTS analysis. The arbitrary value of 1 is referred to dimethyl sulfoxide-treated cells (control) [data are the mean of three separate experiments (error bars, ± SD)]. A greater anti-cancer drug resistance is noted for IGR-N-91 cells and BM metastatic neuroblasts. The difference in viability between SK-N-SH and IGR-N-91 and between PTX and BM is significant and noted (*) if P ≤ 0.05 or (^†) if P ≤ 0.001. B: Cycle analysis was performed in SK-N-SH, IGR-N-91, PTX, and BM cell lines 48 hours after a 10-μmol/L treatment with either VP16, CDDP, or vehicle control (dimethyl sulfoxide). Apoptosis was derived quantitatively by measuring the percentage of sub-G₁ population (inset).

Figure 5.

MycN-mediated induction of MDR1 promoter activity. A: IGR xenograft model (IGR): MYCN and MDR1 transcript levels for MYCN-transfected SK-N-SH neuroblasts and for PTX and BM neuroblasts. Northern blots of 10 μg total from parental SK-N-SH cells, from 0.5 and 1 μg of pMYCN^hu-transfected SK-N-SH cells (left), and from PTX and BM neuroblasts (right), hybridized with ³²P-labeled cDNA probes. Autoradiography of actin probe blotting demonstrated similar loadings. MYCN and MDR1 gene mRNA levels increase in parallel. B: CAT activity of the pMDR1-CAT construct transfected into the SK-N-SH and IGR cells. The SK-N-SH neuroblasts were co-transfected with 0.5 or 1 μg of pMYCN^hu and 10 μg of pMDR1-CAT. The PTX and BM cells were transfected with 10 μg of pMDR1-CAT construct. Controls were performed in SK-N-SH cells transfected with 5 μg of pSVE-CAT construct or co-transfected with 10 μg of pSb1-CAT + 1 μg pMYCN^hu constructs. PSVE and pSb1 constructs are used as positive (1) or negative (2) controlwith strong or weak promoter activity. The experiment was repeated at least twice and a representative transfection is shown. C: Synthetic oligonucleotides are used for gel shift assays. They correspond to the two E-boxes located within the proximal promoter region of the MDR1 gene, either wild-type (MDR1-1 wt and MDR1-2 wt) or mutated (MDR1-2mut) probes. The E-box binding sites are indicated in bold, and mutated bases with asterisks. D: Absence of c-myc protein expression in MYCN-amplified neuroblasts (IGR-N-91), and its presence in MYCN nonamplified neuroblasts (SK-N-SH), as demonstrated by immunoblots of total and nuclear extracts. Max expression is higher in whole extracts of IGR-N-91 cells. E: Electrophoretic mobility shift of oligonucleotide/nuclear protein complexes. Gel shifts were performed using two labeled double-stranded oligonucleotides, MDR1-1 wt and MDR1-2 wt incubated with nuclear extracts from SK-N-SH and IGR-N-91 neuroblasts, without (lanes 1, 3, 5–9, 11) or with (lanes 2, 4, 10) a 25-fold excess of respective competitor. A faint shifted complex is noted with the MDR1-1 probe, and SK-N-SH or IGR-N-91 nuclear extracts (lanes 1 and 5), whereas a stronger shifted complex is evidenced with the MDR1-2 probe (lanes 3, 6–9) with SK-N-SH or IGR-N-91 extracts. No complex is observed in the presence of the MDR1-2 mutated probe or IGR-N-91 nuclear extracts (lane 11).