Poster Presentation Advances in Neuroblastoma Research Congress 2016

Suppression of Multidrug resistance protein 4 inhibits neuroblastoma growth both in vitro and in vivo (#364)

Jayne Murray 1 , Emanuele Valli 1 , Alan Troung 1 , Georgina Eden 1 , Sophie Allan 1 , Hannah Webber 1 , Amanda Tivnan 1 , Alvin Tan 1 , Claudia Flemming 1 , Leanna Cheung 1 , Kimberley Hanssen 1 , Ashleigh Carnegie-Clark 1 , Michelle Ruhle 1 , Michelle Henderson 1 , Glenn M Marshall 1 2 , Denise Yu 1 , Murray D Norris 1 , Jamie I Fletcher 1 , Michelle Haber 1
  1. Children's Cancer Institute, RANDWICK, NSW, Australia
  2. Kids Cancer Centre, Sydney Children's Hospital, Sydney, Australia

Introduction

Multidrug resistance protein 4 (MRP4), encoded by the ABCC4 gene, effluxes a broad range of cytotoxic agents and endogenous signalling molecules (eg cyclic nucleotides and eicosanoids) from cells, and has been proposed as an attractive therapeutic target in cancer. We have previously shown in primary untreated neuroblastoma tumours, that high ABCC4 expression is a powerful independent predictor of poor clinical outcome across multiple patient cohorts [1, 2]. We have also shown that ABCC4 is transcriptionally regulated by the Myc and MYCN oncogenes [1]. MYCN is amplified in approximately 20% of all primary neuroblastomas, and is an important driver of neuroblastoma tumorigenesis. We have now investigated the effect of inhibiting MRP4 on neuroblastoma growth both in vitro and in vivo.

Methods

Human BE(2)-C neuroblastoma cells with doxycycline-inducible expression of MRP4 were developed and characterised for growth and colony forming potential in vitro. Tumour growth was assessed following subcutaneous xenograft in Balb/c nude mice, with administration of doxycycline commencing one week after engraftment. Neuroblastoma-prone TH-MYCN mice were crossed with Abcc4 knock-out mice, and incidence and latency of tumour development studied.

Results

Induction of MRP4 knockdown strongly inhibited BE(2)-C cell growth in vitro and significantly slowed the growth of xenografted neuroblastoma cells. In contrast, however, loss of murine Abcc4 in the TH-MYCN transgenic mouse model did not alter either tumour incidence or the growth rate of established tumours, suggesting differences between human and mouse MRP4 in this context. Knock-out allografts from these mice, when transplanted into Balb/c nude mouse recipients, demonstrated sensitivity to the MRP4 substrate, Irinotecan, compared to wild-type Abcc4 allografts.

Conclusions

Our results suggest that there are significant differences between human and murine MRP4 in their ability to transport physiological substrates. Importantly, loss of MRP4 strongly inhibited growth of human neuroblastoma cells in the absence of any chemotherapeutic drug treatment, highlighting its potential as a therapeutic target in this disease.

  1. Henderson, M.J., et al., ABCC Multidrug Transporters in Childhood Neuroblastoma: Clinical and Biological Effects Independent of Cytotoxic Drug Efflux. Journal of the National Cancer Institute. 103(16): p. 1236-1251.
  2. Norris, M.D., et al., Expression of multidrug transporter MRP4/ABCC4 is a marker of poor prognosis in neuroblastoma and confers resistance to irinotecan in vitro. Molecular Cancer Therapeutics, 2005. 4(4): p. 547-553.