ABCC Multidrug Transporters in Childhood Neuroblastoma: Clinical and Biological Effects Independent of Cytotoxic Drug Efflux

Abstract

BackgroundAlthough the prognostic value of the ATP-binding cassette, subfamily C (ABCC) transporters in childhood neuroblastoma is usually attributed to their role in cytotoxic drug efflux, certain observations have suggested that these multidrug transporters might contribute to the malignant phenotype independent of cytotoxic drug efflux.MethodsA v-myc myelocytomatosis viral related oncogene, neuroblastoma derived (MYCN)–driven transgenic mouse neuroblastoma model was crossed with an Abcc1-deficient mouse strain (658 hMYCN1/−, 205 hMYCN+/1 mice) or, alternatively, treated with the ABCC1 inhibitor, Reversan (n = 20). ABCC genes were suppressed using short interfering RNA or overexpressed by stable transfection in neuroblastoma cell lines BE(2)-C, SH-EP, and SH-SY5Y, which were then assessed for wound closure ability, clonogenic capacity, morphological differentiation, and cell growth. Real-time quantitative polymerase chain reaction was used to examine the clinical significance of ABCC family gene expression in a large prospectively accrued cohort of patients (n = 209) with primary neuroblastomas. Kaplan–Meier survival analysis and Cox regression were used to test for associations with event-free and overall survival. Except where noted, all statistical tests were two-sided.ResultsInhibition of ABCC1 statistically significantly inhibited neuroblastoma development in hMYCN transgenic mice (mean age for palpable tumor: treated mice, 47.2 days; control mice, 41.9 days; hazard ratio [HR] = 9.3, 95% confidence interval [CI] = 2.65 to 32; P < .001). Suppression of ABCC1 in vitro inhibited wound closure (P < .001) and clonogenicity (P = .006); suppression of ABCC4 enhanced morphological differentiation (P < .001) and inhibited cell growth (P < .001). Analysis of 209 neuroblastoma patient tumors revealed that, in contrast with ABCC1 and ABCC4, low rather than high ABCC3 expression was associated with reduced event-free survival (HR of recurrence or death = 2.4, 95% CI = 1.4 to 4.2; P = .001), with 23 of 53 patients with low ABCC3 expression experiencing recurrence or death compared with 31 of 155 patients with high ABCC3. Moreover, overexpression of ABCC3 in vitro inhibited neuroblastoma cell migration (P < .001) and clonogenicity (P = .03). The combined expression of ABCC1, ABCC3, and ABCC4 was associated with patients having an adverse event, such that of the 12 patients with the “poor prognosis” expression pattern, 10 experienced recurrence or death (HR of recurrence or death = 12.3, 95% CI = 6 to 27; P < .001).ConclusionABCC transporters can affect neuroblastoma biology independently of their role in chemotherapeutic drug efflux, enhancing their potential as targets for therapeutic intervention.

Small Molecule Drugs and Targeted Therapies for Neuroblastoma

“…We are currently investigating in more detail precisely how MRP4 contributes to highly malignant neuroblastoma using a range of experimental approaches. In particularly, we have provided strong evidence using siRNA-mediated silencing of MRP4 that this transporter contributes to neuroblastoma biology independently of its role in chemotherapeutic drug efflux (Henderson, Haber et al, 2011). This finding suggests that therapeutic targeting of MRP4 has potential clinical utility for this disease, as well as for other cancers expressing high MRP4 levels.…”

“…However, in terms of MRP1, we have recently shown that this transporter has a more fundamental role in neuroblastoma, than that of simply effluxing cytotoxic drugs. Thus, using genetic and pharmacologic inhibition, we have provided the first direct evidence that MRP1 can contribute to neuroblastoma biology independently of chemotherapeutic drug efflux, thereby enhancing its potential as a target for therapeutic intervention (Henderson, Haber et al, 2011). Importantly, in this study, treatment of neuroblastoma-prone hMYCN transgenic mice with Reversan in the absence of chemotherapeutic agents, led to a significant delay in tumour progression Although available evidence indicates that Reversan is not a substrate for MRP1, its exact mechanism of action as well as related pyrazolopyrimidines is currently unknown.…”

Screening to Identify Multidrug Resistance-Associated Protein Inhibitors with Neuroblastoma-Selective Cytotoxicity

“…23,24) High expression of MRP1 or MRP4 was reported to be associated with a poor prognosis of neuroblastoma. 17,25) Henderson et al also proposed that MRPs may be involved in exacerbation of neuroblastoma via unknown mechanisms in humans, and administration of the MRP1-selective inhibitor reversan (a lead compound of pyrazolopyrimidines, a prominent structural class of potent MRP1 inhibitors) suppressed growth of implanted neuroblastoma in mice. 17) MRP3 mediates efflux transport of various endogenous compounds and metabolites, including leukotriene C4, glycocholic acid and estradiol glucuronide, 26) from intracellular to extracellular space.…”

“…cDNA was synthesized with oligo(dT) [12][13][14][15][16][17][18] primer, deoxynucleotide triphosphate mix, RT buffer and MultiScribe Reverse Transcriptase, and amplified on a Mx3005P (Agilent Technologies, Santa Clara, CA, U.S.A.) in a reaction mixture containing cDNA with relevant sense and antisense primers (Table 1), and THUNDERBIRD SYBR qPCR Mix. PCR reactions were initiated by template denaturation at 95°C for 15 min, followed by 40 cycles of amplification (denaturation at 95°C for 10 s, and primer annealing and extension at 60°C for 30 s).…”

“…Inhibition of some SLC transporters has been reported to have anticancer effects. 12,14,15,17) On the other hand, most work on ABC transporters has been focused on ABC transporters that mainly recognize xenobiotics and mediate elimination of anticancer drugs used in chemotherapy. We were interested in the possibility that inhibitors of ABC xenobiotic transporters might show selective cytotoxicity towards neuroblastoma, with minimal neurotoxicity.…”