title: ABCC multidrug transporters in childhood neuroblastoma: clinical and biological effects independent of cytotoxic drug efflux.
abstract: BACKGROUND
  Although 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.
  
  
  METHODS
  A v-myc myelocytomatosis viral related oncogene, neuroblastoma derived (MYCN)-driven transgenic mouse neuroblastoma model was crossed with an Abcc1-deficient mouse strain (658 hMYCN(1/-), 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.
  
  
  RESULTS
  Inhibition 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).
  
  
  CONCLUSIONS
  ABCC transporters can affect neuroblastoma biology independently of their role in chemotherapeutic drug efflux, enhancing their potential as targets for therapeutic intervention.
authors.affiliation: Experimental Therapeutics and Molecular Diagnostics Program, Children's Cancer Institute Australia for Medical Research, Lowy Cancer Research Centre, University of New South Wales, P.O. Box 81, Randwick, NSW 2031, Australia.
authors.forename: Michelle J
authors.initials: MJ
authors.lastname: Henderson
authors.forename: Michelle
authors.initials: M
authors.lastname: Haber
authors.forename: Antonio
authors.initials: A
authors.lastname: Porro
authors.forename: Marcia A
authors.initials: MA
authors.lastname: Munoz
authors.forename: Nunzio
authors.initials: N
authors.lastname: Iraci
authors.forename: Chengyuan
authors.initials: C
authors.lastname: Xue
authors.forename: Jayne
authors.initials: J
authors.lastname: Murray
authors.forename: Claudia L
authors.initials: CL
authors.lastname: Flemming
authors.forename: Janice
authors.initials: J
authors.lastname: Smith
authors.forename: Jamie I
authors.initials: JI
authors.lastname: Fletcher
authors.forename: Samuele
authors.initials: S
authors.lastname: Gherardi
authors.forename: Chin-Kiat
authors.initials: CK
authors.lastname: Kwek
authors.forename: Amanda J
authors.initials: AJ
authors.lastname: Russell
authors.forename: Emanuele
authors.initials: E
authors.lastname: Valli
authors.forename: Wendy B
authors.initials: WB
authors.lastname: London
authors.forename: Allen B
authors.initials: AB
authors.lastname: Buxton
authors.forename: Lesley J
authors.initials: LJ
authors.lastname: Ashton
authors.forename: Alan C
authors.initials: AC
authors.lastname: Sartorelli
authors.forename: Susan L
authors.initials: SL
authors.lastname: Cohn
authors.forename: Manfred
authors.initials: M
authors.lastname: Schwab
authors.forename: Glenn M
authors.initials: GM
authors.lastname: Marshall
authors.forename: Giovanni
authors.initials: G
authors.lastname: Perini
authors.forename: Murray D
authors.initials: MD
authors.lastname: Norris
mesh_terms: D000293:Adolescent; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D015153:Blotting, Western; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D002465:Cell Movement; D002648:Child; D002675:Child, Preschool; D004195:Disease Models, Animal; D018572:Disease-Free Survival; D015536:Down-Regulation; D018432:Drug Resistance, Multiple; D019008:Drug Resistance, Neoplasm; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D020868:Gene Silencing; D006801:Humans; D007223:Infant; D053208:Kaplan-Meier Estimate; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000071447:N-Myc Proto-Oncogene Protein; D009447:Neuroblastoma / Q000188:drug therapy* / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism; D016017:Odds Ratio; D015513:Oncogene Proteins / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011237:Predictive Value of Tests; D011379:Prognosis; D016016:Proportional Hazards Models; D011446:Prospective Studies; D011720:Pyrazoles / Q000494:pharmacology*; D011743:Pyrimidines / Q000494:pharmacology*; D034741:RNA, Small Interfering / Q000378:metabolism; D012008:Recurrence; D013997:Time Factors; D014162:Transfection; D015854:Up-Regulation; D055815:Young Adult
chemical_list: D000970:Antineoplastic Agents; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D000071447:N-Myc Proto-Oncogene Protein; D009687:Nuclear Proteins; D015513:Oncogene Proteins; D011720:Pyrazoles; D011743:Pyrimidines; D034741:RNA, Small Interfering; C073492:ABCC4 protein, human; C459801:MYCN protein, human; C543413:reversan; C114462:multidrug resistance-associated protein 3; C421109:multidrug resistance-associated protein 1