Date of Award

12-2007

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Pharmaceutical Sciences

Research Advisor

Clinton Stewart, Pharm.D.

Committee

Mary Danks, Ph.D. Bernd Meibohm, Ph.D. Victor Santana, M.D. John Schuetz, Ph.D.

Keywords

Neuroblastoma, topotecan, irinotecan, ABC transporter, multidrug resistance, MRP4, Pgp, RNAi, shRNA, siRNA, immunohistochemistry

Abstract

High-risk neuroblastoma presents a significant therapeutic challenge because the 5-year survival rate remains less than 30% despite the use of surgery, multi-agent chemotherapy, radiation, and autologous bone marrow transplant. Novel therapeutic modalities are under development. The camptothecin analogs topotecan and irinotecan have been identified as successful cytotoxic agents. For topotecan, pharmacokinetically guided dosing to achieve a systemic exposure associated with preclinical anti-tumor activity in neuroblastoma xenograft models is feasible and has elicited favorable responses in children with high-risk neuroblastoma. However, some children with high-risk disease did not respond to the putatively effective topotecan systemic exposure. These children represent a subset of the disease intrinsically resistant to topotecan. Furthermore, mRNA expression of the adenosine triphosphate (ATP)-binding cassette (ABC) transporters P-glycoprotein (Pgp) and multidrug resistance associated protein 1 (MRP1), which efflux many drugs used in neuroblastoma therapy, has been implicated in poor outcome in neuroblastoma. Therefore, the purpose of our studies was to determine the role of ABC transport protein expression in neuroblastoma resistance to the camptothecin analogs topotecan and irinotecan.

Initially studies focused on determining the expression of ABC transporters for which the camptothecin analogs are substrates in neuroblastoma cell lines. By western blot analysis we demonstrated MRP4 and Pgp expression in neuroblastoma cell lines relatively resistant to topotecan (e.g., NB1691), but not in cell lines sensitive to topotecan (e.g., NB1643). In contrast, MRP1, MRP2, and breast cancer resistance protein (BCRP) expression did not discriminate between sensitive and resistant cell lines. To determine the functional contribution of both MRP4 and Pgp in neuroblastoma, we used RNA interference (RNAi) to silence MRP4 and Pgp expression in NB1691. Long term, stable expression of retroviral vector mediated short hairpin RNA (shRNA) reduced MRP4 and Pgp expression. Isogenic cell lines with reduced expression of MRP4 and Pgp exhibited an increase in sensitivity to both topotecan and SN-38, the active moiety of the prodrug irinotecan. In addition, we overexpressed MRP4 in NB1643, which resulted in increased topotecan resistance.

The NB1691 cell lines with reduced MRP4 expression were subsequently transplanted as xenografts into severe combined immunodeficiency (SCID) mice to determine the effect of MRP4 expression on the in vivo response to topotecan. Unexpectedly, MRP4 silencing did not persist in vivo, and none of the xenograft models responded to topotecan. However, MRP4 expression was associated with failure to respond to topotecan, supporting the hypothesis that MRP4 mediates resistance to topotecan.

Finally, we determined the ABC transporter expression profile in primary tumor specimens from patients with high-risk neuroblastoma who were treated with pharmacokinetically guided topotecan. Of the 14 specimens studied, MRP4 was expressed in 2 samples, and Pgp was expressed in 4 samples. BCRP was not expressed in any of the neuroblastoma cell lines in vitro, but immunohistochemical analysis demonstrated BCRP expression in nine primary neuroblastoma samples. Although we predicted that MRP4 and/or Pgp expression would be associated with failure to respond to topotecan, results of immunohistochemical analysis did not demonstrate such an association.

The results of the in vitro studies demonstrate that MRP4 and Pgp confer resistance to topotecan and SN-38. In the xenograft studies, MRP4 expression was associated with failure to respond to topotecan. However, this phenotype was not recapitulated in children treated with topotecan. These results may be confounded by small sample size and timing of sample acquisition. Further investigation of the role of ABC transporters in children with neuroblastoma who receive either topotecan or irinotecan may be warranted. In addition to the camptothecin analogs, patients will receive other drugs effluxed by the ABC transporters (e.g., doxorubicin, vincristine, etoposide, cyclophosphamide). Therefore, analyzing ABC transporter expression by immunohistochemistry in diagnostic tumor specimens may help to select agents not subject to efflux by ABC transporters expressed in the tumor. However, eliminating drugs effluxed by ABC transporters from the treatment regimen creates a potential gap in therapy and may reduce drug intensity. Therefore, further rational design and development of drugs that evade ABC transporter-mediated efflux, and potentially other resistance mechanisms in neuroblastoma, is also warranted.

DOI

10.21007/etd.cghs.2007.0325

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