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The role of the multidrug resistance-associated protein 1 gene in neuroblastoma biology and clinical outcome
Cancer Letters 228 (2005) 241–246 www.elsevier.com/locate/canlet
The role of the multidrug resistance-associated protein 1 gene in neuroblastoma biology and clinical outcome Marina Pajica, Murray D. Norrisa, S.L. Cohnb, Michelle Habera,* a
Children’s Cancer Institute Australia for Medical Research, P.O. Box 81 Randwick, Sydney, NSW 2031, Australia b Department of Pediatrics, Northwestern University, Feinberg School Of Medicine, Chicago IL, USA Received 15 December 2004; accepted 12 January 2005
Abstract Multidrug resistance is a major obstacle to cancer treatment and leads to poor prognosis for the patient. Multidrug resistanceassociated protein 1 (MRP1) can confer drug resistance in vitro and MRP1 may play a role in the development of drug resistance in several cancers including acute myeloid leukaemia, small cell lung cancer, T-cell leukaemia and neuroblastoma. The majority of patients with neuroblastoma present with widely disseminated disease at diagnosis and despite intensive treatment, the prognosis for such patients is dismal. There is increasing evidence for the involvement of the MYCN oncogene, and its down-stream target, MRP1, in the development of multidrug resistance in neuroblastoma. Given the importance of MRP1 overexpression in neuroblastoma, MRP1 inhibition may be a clinically relevant approach to improving patient outcome in this disease. q 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Neuroblastoma; MRP1; ABCC1; MYCN; Drug resistance; Cytotoxic
1. Introduction Although significant advances have been made in the treatment of various types of cancer, drug resistance remains a major clinical problem in highrisk neuroblastoma where it often leads to limited therapeutic options and poor patient outcome, and is associated with an overall survival rate of only 30% [1]. A major contributing factor to the chemotherapeutic treatment failure of this disease is
* Corresponding author. Tel.: C61 2 9382 1821; fax: C61 2 9382 1850. E-mail address: [email protected] (M. Haber).
the development of resistance to a broad range of cytotoxic drugs. Better understanding of the drug resistance mechanisms is needed in order to improve the current chemotherapy regimens. Multidrug resistance (MDR) is characterised by cross-resistance to a variety of both structurally and pharmacologically unrelated drugs and it can be either intrinsic or acquired. It is frequently associated with overexpression of one or more MDR transporter proteins, which function as ATP-dependent pumps to extrude an extraordinarily diverse array of endo- and xenobiotics out of the cell. The best characterised multidrug transporters are the ATP-binding cassette (ABC) gene superfamily of which at least six are
0304-3835/$ - see front matter q 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2005.01.060
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associated with transport of anticancer drugs [2]. They include P-glycoprotein (Pgp) and the Multidrug Resistance-associated Protein (MRP) family. Of all the MRP family members, MRP1 has been most closely linked to the development of clinical MDR for several types of cancer, including neuroblastoma. This review highlights the available data on the role of MRP1 in neuroblastoma, and provides evidence that inhibition of this transporter may be a novel approach to improving patient therapy in this disease.
2. Overview of drug resistance conferred by MRP1 First cloned in 1992 [3], the MRP1 gene was found to encode a 190kDa membrane-bound protein whose overexpression conferred MDR in a range of in vitro systems. In common with the well-characterised multidrug transporter Pgp, MRP1 can confer resistance to a broad range of natural product drugs including Vinca alkaloids, anthracyclines and epipodophyllotoxins. In contrast to Pgp, however, MRP1 transports a broader range of xenobiotics, including heavy metals, and its physiological substrates include estrone sulfate and leukotriene C4. Moreover, it effluxes drugs either conjugated to glutathione (GSH) or by a direct GSH co-transport mechanism [4]. Therefore, although the drug resistance phenotype of MRP1 overlaps that of P-glycoprotein it is still nevertheless quite distinct. 2.1. Physiological and pathological roles of MRP1 Although the precise physiological role of MRP1 has not been defined, this transporter protein may be important in protecting cells against oxidative stress [5]. MRP1 knockout mice demonstrate hypersensitivity to anticancer agents and an impaired response to inflammatory stimuli that can be linked to decreased secretion of leukotriene C4 from mast cells [6]. Importantly, these animals are healthy and fertile indicating that this transporter is not essential to life [5]. As is the case with other transporters, direct evidence for a causative role of MRP1 in clinical drug resistance has to date been difficult to prove. Nevertheless, MRP1 has been clearly implicated as having a role in the clinical drug resistance behaviour of several cancers including small cell lung cancer, acute
myeloid leukemia, T-cell leukemia (reviewed in [4]) and increasingly, in childhood neuroblastoma [7].
3. Regulation of the MRP1 gene The MRP1 gene, located on chromosome 16p3.1, is regulated by numerous transcription factors, including GC elements within the MRP1 promoter (K91–C103) essential for activity, Sp1, YB-1, AP-1 and WT-1 (reviewed in [2]). Transcription of the MRP1 promoter can be repressed by functional p53 [8], and p53 may also play a role in regulation of MRP1 expression in colorectal cancer [9]. Of particular relevance to neuroblastoma, there is increasing evidence that the MYCN oncogene, which is a clinical determinant of neuroblastoma outcome, may regulate MRP1 expression in this disease [10]. 3.1. MYCN regulation of MRP1 Amplification of the MYCN oncogene in neuroblastoma is one of the most powerful prognostic indicators for this disease. It is often associated with rapid tumour progression, advanced stage disease and poor outcome [11]. MYCN has been shown to critically contribute to the malignant phenotype of neuroblastoma in vitro and is involved in growth and differentiation of neuroblastoma cells [12,13], however the specific mechanisms by which this occurs remain mostly unknown. The MYCN gene encodes a 60–63 kDa nuclear phosphoprotein that contains a basic helix-loop helix/leucine zipper (bHLH-LZ) motif [14]. In order to activate transcription the MYCN protein dimerizes to MAX, another bHLH-LZ protein [15,16]. We have recently obtained direct evidence of MYCN-mediated regulation of the MRP1 gene, from studies of the MRP1 promoter [10]. Specifically, using luciferase constructs, increased levels of MRP1 promoter activity were demonstrated in neuroblastoma cell lines with stable overexpression of MYCN or following tetracycline-regulated induction. Deletion constructs and electrophoretic mobility shift assays showed that MYCN regulates the MRP1 gene through interaction with a putative E-box element (E-box 3) and other cis-acting factors in the MRP1 promoter (Fig. 1). Consequently, the enhanced levels of MRP1
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Fig. 1. A simplified diagram of MYCN-mediated regulation of MRP1 signalling. MYCN binds to specific sequences in the MRP1promoter, primarily E-box 3, inducing transcription of the MRP1 gene, and resulting in increased MRP1-mediated drug resistance.
expression result in increased drug resistance suggesting that MRP1 may be a MYCN target gene involved in the MDR phenotype of neuroblastoma tumours [10] (Fig. 1).
4. MRP1 in primary neuroblastoma In 1994, MRP1 expression in primary human neuroblastoma was first shown to directly correlate with the expression of the MYCN oncogene [17]. Furthermore in the same study, treatment of neuroblastoma cell lines with retinoic acid resulted in a coordinated down-regulation of both the MYCN and MRP1 genes. We and others have since confirmed that high expression of the MRP1 gene in neuroblastoma directly correlates with amplification and overexpression of the MYCN gene [7,18–20]. In a retrospective study of 60 primary neuroblastoma specimens, high MRP1 expression was strongly associated with both reduced overall and event-free survival, and MRP1 expression was also prognostic of outcome in MYCN non-amplified tumour samples and in patients with localized disease [7]. Importantly, when outcome was adjusted for the effect of MYCN amplification, MRP1 expression remained a significant prognostic indicator of both poor survival and event-free survival [7].
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We have now confirmed these findings in a prospective analysis of MRP1 expression in a cohort of 209 primary untreated neuroblastoma samples from patients enrolled on POG biology protocol 9047. High levels of MRP1 were highly predictive of poor patient outcome. Following adjustment for MYCN amplification and other prognostic factors using multivariate analysis, MRP1 expression remained a significant prognostic indicator for both event-free and overall survival. In contrast to these results, MDR1 expression demonstrated no prognostic significance (manuscript in preparation). Thus, the results of the prospective study confirm our earlier findings and support a clinically relevant role for MRP1 gene expression in drug refractory neuroblastoma. P-glycoprotein on the other hand appears to have a more limited role in the development of drug resistance in neuroblastoma [21– 25]. Another study [26] has reported a significant association between high MRP1 expression and MYCN amplification and overexpression and poor outcome in the overall study group, as well as advanced disease. However in stages I, II and IV-S (early, non-aggressive) tumours, MRP1 expression did not influence overall survival. Patients with these tumours usually do well after treatment, suggesting that the characteristics of these tumours are inherently different in comparison with the advanced stages of neuroblastoma. Further evidence of a role for MRP1 in neuroblastoma has been provided in a recent study examining MRP1, Pgp and LRP protein levels in a cohort of 70 primary untreated tumours [27]. Of these three drug resistance proteins, only MRP1 was significantly associated with patient outcome.
5. In vitro and in vivo MRP1 drug resistance models Numerous studies have demonstrated that transfection of the MRP1 gene confers a multi-drug resistant phenotype in a variety of tumour cell types [5]. Moreover, in considering a role for MRP1 in clinical drug resistance, a recent study has investigated the contribution of basal levels of MRP1, as well as of Pgp, to drug sensitivity, by examining the response to cytotoxic drugs of mouse cells in which these genes had been made non-functional through targeted disruption [28]. Although it was found that both
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transporters markedly contributed to drug resistance, MRP1 disruption conferred significant hypersensitivity to an even broader range of drugs than Pgp. Given the clear association between high MRP1 expression and the malignant and drug-resistant phenotype of neuroblastoma both in vitro and in clinical neuroblastoma, MRP1 expression appears to be a highly relevant clinical parameter in this disease. As such, MRP1 is potentially an important target for reversing chemotherapy resistance in neuroblastoma patients. 5.1. In vitro drug resistance Antisense technology has been utilized successfully to demonstrate the potential of inhibiting MRP1 gene expression in neuroblastoma cells either directly, or through inhibition of the MYCN gene which in turn resulted in reduced expression of its target, MRP1. In neuroblastoma cell lines with constitutively high levels of MYCN expression, significant inhibition of expression of both MYCN and its target gene MRP1 were demonstrated following stable transfection with MYCN antisense RNA constructs [29]. These findings were confirmed at the protein level, and importantly, decreased MRP1 expression in these MYCN antisense transfectants was associated with a marked increase in sensitivity of these cells to a range of cytotoxic drugs known to be MRP1 substrates, but not to non-MRP1 substrate drugs. In addition to influencing cytotoxic drug response, inhibition of MRP1 appears to have additional desirable effects on neuroblastoma cells. Thus, transfection of MRP1-overexpressing human neuroblastoma BE(2)-C cells with MRP1-antisense constructs was shown to induce differentiation and enhance apoptosis in this system [30], suggesting that MRP1 overexpression in aggressive neuroblastoma may have a role in preventing neuritic differentiation and cell death in the tumour. 5.2. MRP1 and in vivo drug resistance in neuroblastoma The recently developed MYCN-transgenic mouse represents a valuable animal model of aggressive neuroblastoma [31]. In these animals, human MYCN (hMYCN) oncogene expression was targeted to neural crest cells with the use of a rat tyrosine hydroxylase
promoter. Importantly, this model closely mirrors the human disease in terms of major molecular, biological and cytogenetic features, including chromosome 1p36 deletion, gain of 17q [32,33], and tumour-specific increase in the copy number of the hMYCN gene [34]. Consistent with the findings in primary neuroblastoma tumour samples, we have found a significant correlation between hMYCN and MRP1 expression in the tumours of these mice [35]. In the same study we found no association between expression of hMYCN transgene and mdr1a or mdr1b, the murine homologues of the human MDR1 gene. Continuous treatment of MYCN-transgenic mice with MYCN phosphorothioate antisense oligonucleotides via micro-osmotic pumps resulted in marked downregulation of both expression of hMYCN and its target genes, including MRP1 [35]. By crossing MYCN-transgenic mice with MRP1knockout mice [36], we have generated progeny with varying levels of MRP1 in their neuroblastoma tumours. When tumours lacking MRP1 were xenografted into nude mice, significantly improved survival of host mice was observed following treatment with MRP1 substrate cytotoxic drugs, by comparison with tumours wild-type for the MRP1 gene. In contrast, there was no difference in survival when mice were treated with non-MRP1 substrate drugs (manuscript in preparation). Independently, administration of MRP1-antisense oligonucleotides in an in vivo mouse-human xenograft model of neuroblastoma has been shown to result in significant downregulation of MRP1 levels in the tumour, associated with a dramatic increase in apoptotic cell death [37]. Furthermore, downregulation of MRP1 resulted in significant chemosensitisation of tumour xenografts to a clinically used chemotherapy agent etoposide, a known MRP1 substrate [37]. Thus, there is accumulating evidence that MRP1 plays an important role in the clinical drug resistance of neuroblastoma, and that MRP1 modulation may be a viable approach to improving patient response in this disease. 5.3. MRP1 inhibitors PgP and MRP1 have been actively pursued as targets for therapeutic suppression, since it is widely believed that safe and potent inhibitors of these
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proteins would be clinically efficacious in increasing the sensitivity of drug-resistant cancers to chemotherapy. Several MRP1-specific reversing agents have been identified in vitro and these include the leukotriene C4 antagonist MK571, the benzothipene LY329146, the dihydropiridine analogue NIK250, the quinolone antibiotic difloxacin, and the organic acids, sulfinpyrazone, benzbromarone and probenecid [4]. However, to date there are few specific clinically relevant MRP1 modulators and therefore this remains an important and ongoing area of research.
6. Conclusion The study of the role of MRP1 in neuroblastoma has provided important insights into the drug refractory behaviour of this disease, and available evidence indicates that elucidating the pathways involved in MRP1 signalling will create new approaches to improving patient outcome in this disease. Screening of small molecule chemical libraries to identify specific inhibitors of the MRP1 transporter is currently underway in our laboratory and we have identified compounds that appear to be highly effective at enhancing sensitivity to MRP1-substrate drugs both in vitro and in vivo. Such compounds may potentially provide an effective new treatment approach for neuroblastoma and other malignancies in which MRP1 plays a clinically relevant role.
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The role of the multidrug resistance-associated protein 1 gene in neuroblastoma biology and clinical outcome Marina Pajica, Murray D. Norrisa, S.L. Cohnb, Michelle Habera,* a
Children’s Cancer Institute Australia for Medical Research, P.O. Box 81 Randwick, Sydney, NSW 2031, Australia b Department of Pediatrics, Northwestern University, Feinberg School Of Medicine, Chicago IL, USA Received 15 December 2004; accepted 12 January 2005
Abstract Multidrug resistance is a major obstacle to cancer treatment and leads to poor prognosis for the patient. Multidrug resistanceassociated protein 1 (MRP1) can confer drug resistance in vitro and MRP1 may play a role in the development of drug resistance in several cancers including acute myeloid leukaemia, small cell lung cancer, T-cell leukaemia and neuroblastoma. The majority of patients with neuroblastoma present with widely disseminated disease at diagnosis and despite intensive treatment, the prognosis for such patients is dismal. There is increasing evidence for the involvement of the MYCN oncogene, and its down-stream target, MRP1, in the development of multidrug resistance in neuroblastoma. Given the importance of MRP1 overexpression in neuroblastoma, MRP1 inhibition may be a clinically relevant approach to improving patient outcome in this disease. q 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Neuroblastoma; MRP1; ABCC1; MYCN; Drug resistance; Cytotoxic
1. Introduction Although significant advances have been made in the treatment of various types of cancer, drug resistance remains a major clinical problem in highrisk neuroblastoma where it often leads to limited therapeutic options and poor patient outcome, and is associated with an overall survival rate of only 30% [1]. A major contributing factor to the chemotherapeutic treatment failure of this disease is
* Corresponding author. Tel.: C61 2 9382 1821; fax: C61 2 9382 1850. E-mail address: [email protected] (M. Haber).
the development of resistance to a broad range of cytotoxic drugs. Better understanding of the drug resistance mechanisms is needed in order to improve the current chemotherapy regimens. Multidrug resistance (MDR) is characterised by cross-resistance to a variety of both structurally and pharmacologically unrelated drugs and it can be either intrinsic or acquired. It is frequently associated with overexpression of one or more MDR transporter proteins, which function as ATP-dependent pumps to extrude an extraordinarily diverse array of endo- and xenobiotics out of the cell. The best characterised multidrug transporters are the ATP-binding cassette (ABC) gene superfamily of which at least six are
0304-3835/$ - see front matter q 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2005.01.060
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associated with transport of anticancer drugs [2]. They include P-glycoprotein (Pgp) and the Multidrug Resistance-associated Protein (MRP) family. Of all the MRP family members, MRP1 has been most closely linked to the development of clinical MDR for several types of cancer, including neuroblastoma. This review highlights the available data on the role of MRP1 in neuroblastoma, and provides evidence that inhibition of this transporter may be a novel approach to improving patient therapy in this disease.
2. Overview of drug resistance conferred by MRP1 First cloned in 1992 [3], the MRP1 gene was found to encode a 190kDa membrane-bound protein whose overexpression conferred MDR in a range of in vitro systems. In common with the well-characterised multidrug transporter Pgp, MRP1 can confer resistance to a broad range of natural product drugs including Vinca alkaloids, anthracyclines and epipodophyllotoxins. In contrast to Pgp, however, MRP1 transports a broader range of xenobiotics, including heavy metals, and its physiological substrates include estrone sulfate and leukotriene C4. Moreover, it effluxes drugs either conjugated to glutathione (GSH) or by a direct GSH co-transport mechanism [4]. Therefore, although the drug resistance phenotype of MRP1 overlaps that of P-glycoprotein it is still nevertheless quite distinct. 2.1. Physiological and pathological roles of MRP1 Although the precise physiological role of MRP1 has not been defined, this transporter protein may be important in protecting cells against oxidative stress [5]. MRP1 knockout mice demonstrate hypersensitivity to anticancer agents and an impaired response to inflammatory stimuli that can be linked to decreased secretion of leukotriene C4 from mast cells [6]. Importantly, these animals are healthy and fertile indicating that this transporter is not essential to life [5]. As is the case with other transporters, direct evidence for a causative role of MRP1 in clinical drug resistance has to date been difficult to prove. Nevertheless, MRP1 has been clearly implicated as having a role in the clinical drug resistance behaviour of several cancers including small cell lung cancer, acute
myeloid leukemia, T-cell leukemia (reviewed in [4]) and increasingly, in childhood neuroblastoma [7].
3. Regulation of the MRP1 gene The MRP1 gene, located on chromosome 16p3.1, is regulated by numerous transcription factors, including GC elements within the MRP1 promoter (K91–C103) essential for activity, Sp1, YB-1, AP-1 and WT-1 (reviewed in [2]). Transcription of the MRP1 promoter can be repressed by functional p53 [8], and p53 may also play a role in regulation of MRP1 expression in colorectal cancer [9]. Of particular relevance to neuroblastoma, there is increasing evidence that the MYCN oncogene, which is a clinical determinant of neuroblastoma outcome, may regulate MRP1 expression in this disease [10]. 3.1. MYCN regulation of MRP1 Amplification of the MYCN oncogene in neuroblastoma is one of the most powerful prognostic indicators for this disease. It is often associated with rapid tumour progression, advanced stage disease and poor outcome [11]. MYCN has been shown to critically contribute to the malignant phenotype of neuroblastoma in vitro and is involved in growth and differentiation of neuroblastoma cells [12,13], however the specific mechanisms by which this occurs remain mostly unknown. The MYCN gene encodes a 60–63 kDa nuclear phosphoprotein that contains a basic helix-loop helix/leucine zipper (bHLH-LZ) motif [14]. In order to activate transcription the MYCN protein dimerizes to MAX, another bHLH-LZ protein [15,16]. We have recently obtained direct evidence of MYCN-mediated regulation of the MRP1 gene, from studies of the MRP1 promoter [10]. Specifically, using luciferase constructs, increased levels of MRP1 promoter activity were demonstrated in neuroblastoma cell lines with stable overexpression of MYCN or following tetracycline-regulated induction. Deletion constructs and electrophoretic mobility shift assays showed that MYCN regulates the MRP1 gene through interaction with a putative E-box element (E-box 3) and other cis-acting factors in the MRP1 promoter (Fig. 1). Consequently, the enhanced levels of MRP1
M. Pajic et al. / Cancer Letters 228 (2005) 241–246
Fig. 1. A simplified diagram of MYCN-mediated regulation of MRP1 signalling. MYCN binds to specific sequences in the MRP1promoter, primarily E-box 3, inducing transcription of the MRP1 gene, and resulting in increased MRP1-mediated drug resistance.
expression result in increased drug resistance suggesting that MRP1 may be a MYCN target gene involved in the MDR phenotype of neuroblastoma tumours [10] (Fig. 1).
4. MRP1 in primary neuroblastoma In 1994, MRP1 expression in primary human neuroblastoma was first shown to directly correlate with the expression of the MYCN oncogene [17]. Furthermore in the same study, treatment of neuroblastoma cell lines with retinoic acid resulted in a coordinated down-regulation of both the MYCN and MRP1 genes. We and others have since confirmed that high expression of the MRP1 gene in neuroblastoma directly correlates with amplification and overexpression of the MYCN gene [7,18–20]. In a retrospective study of 60 primary neuroblastoma specimens, high MRP1 expression was strongly associated with both reduced overall and event-free survival, and MRP1 expression was also prognostic of outcome in MYCN non-amplified tumour samples and in patients with localized disease [7]. Importantly, when outcome was adjusted for the effect of MYCN amplification, MRP1 expression remained a significant prognostic indicator of both poor survival and event-free survival [7].
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We have now confirmed these findings in a prospective analysis of MRP1 expression in a cohort of 209 primary untreated neuroblastoma samples from patients enrolled on POG biology protocol 9047. High levels of MRP1 were highly predictive of poor patient outcome. Following adjustment for MYCN amplification and other prognostic factors using multivariate analysis, MRP1 expression remained a significant prognostic indicator for both event-free and overall survival. In contrast to these results, MDR1 expression demonstrated no prognostic significance (manuscript in preparation). Thus, the results of the prospective study confirm our earlier findings and support a clinically relevant role for MRP1 gene expression in drug refractory neuroblastoma. P-glycoprotein on the other hand appears to have a more limited role in the development of drug resistance in neuroblastoma [21– 25]. Another study [26] has reported a significant association between high MRP1 expression and MYCN amplification and overexpression and poor outcome in the overall study group, as well as advanced disease. However in stages I, II and IV-S (early, non-aggressive) tumours, MRP1 expression did not influence overall survival. Patients with these tumours usually do well after treatment, suggesting that the characteristics of these tumours are inherently different in comparison with the advanced stages of neuroblastoma. Further evidence of a role for MRP1 in neuroblastoma has been provided in a recent study examining MRP1, Pgp and LRP protein levels in a cohort of 70 primary untreated tumours [27]. Of these three drug resistance proteins, only MRP1 was significantly associated with patient outcome.
5. In vitro and in vivo MRP1 drug resistance models Numerous studies have demonstrated that transfection of the MRP1 gene confers a multi-drug resistant phenotype in a variety of tumour cell types [5]. Moreover, in considering a role for MRP1 in clinical drug resistance, a recent study has investigated the contribution of basal levels of MRP1, as well as of Pgp, to drug sensitivity, by examining the response to cytotoxic drugs of mouse cells in which these genes had been made non-functional through targeted disruption [28]. Although it was found that both
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transporters markedly contributed to drug resistance, MRP1 disruption conferred significant hypersensitivity to an even broader range of drugs than Pgp. Given the clear association between high MRP1 expression and the malignant and drug-resistant phenotype of neuroblastoma both in vitro and in clinical neuroblastoma, MRP1 expression appears to be a highly relevant clinical parameter in this disease. As such, MRP1 is potentially an important target for reversing chemotherapy resistance in neuroblastoma patients. 5.1. In vitro drug resistance Antisense technology has been utilized successfully to demonstrate the potential of inhibiting MRP1 gene expression in neuroblastoma cells either directly, or through inhibition of the MYCN gene which in turn resulted in reduced expression of its target, MRP1. In neuroblastoma cell lines with constitutively high levels of MYCN expression, significant inhibition of expression of both MYCN and its target gene MRP1 were demonstrated following stable transfection with MYCN antisense RNA constructs [29]. These findings were confirmed at the protein level, and importantly, decreased MRP1 expression in these MYCN antisense transfectants was associated with a marked increase in sensitivity of these cells to a range of cytotoxic drugs known to be MRP1 substrates, but not to non-MRP1 substrate drugs. In addition to influencing cytotoxic drug response, inhibition of MRP1 appears to have additional desirable effects on neuroblastoma cells. Thus, transfection of MRP1-overexpressing human neuroblastoma BE(2)-C cells with MRP1-antisense constructs was shown to induce differentiation and enhance apoptosis in this system [30], suggesting that MRP1 overexpression in aggressive neuroblastoma may have a role in preventing neuritic differentiation and cell death in the tumour. 5.2. MRP1 and in vivo drug resistance in neuroblastoma The recently developed MYCN-transgenic mouse represents a valuable animal model of aggressive neuroblastoma [31]. In these animals, human MYCN (hMYCN) oncogene expression was targeted to neural crest cells with the use of a rat tyrosine hydroxylase
promoter. Importantly, this model closely mirrors the human disease in terms of major molecular, biological and cytogenetic features, including chromosome 1p36 deletion, gain of 17q [32,33], and tumour-specific increase in the copy number of the hMYCN gene [34]. Consistent with the findings in primary neuroblastoma tumour samples, we have found a significant correlation between hMYCN and MRP1 expression in the tumours of these mice [35]. In the same study we found no association between expression of hMYCN transgene and mdr1a or mdr1b, the murine homologues of the human MDR1 gene. Continuous treatment of MYCN-transgenic mice with MYCN phosphorothioate antisense oligonucleotides via micro-osmotic pumps resulted in marked downregulation of both expression of hMYCN and its target genes, including MRP1 [35]. By crossing MYCN-transgenic mice with MRP1knockout mice [36], we have generated progeny with varying levels of MRP1 in their neuroblastoma tumours. When tumours lacking MRP1 were xenografted into nude mice, significantly improved survival of host mice was observed following treatment with MRP1 substrate cytotoxic drugs, by comparison with tumours wild-type for the MRP1 gene. In contrast, there was no difference in survival when mice were treated with non-MRP1 substrate drugs (manuscript in preparation). Independently, administration of MRP1-antisense oligonucleotides in an in vivo mouse-human xenograft model of neuroblastoma has been shown to result in significant downregulation of MRP1 levels in the tumour, associated with a dramatic increase in apoptotic cell death [37]. Furthermore, downregulation of MRP1 resulted in significant chemosensitisation of tumour xenografts to a clinically used chemotherapy agent etoposide, a known MRP1 substrate [37]. Thus, there is accumulating evidence that MRP1 plays an important role in the clinical drug resistance of neuroblastoma, and that MRP1 modulation may be a viable approach to improving patient response in this disease. 5.3. MRP1 inhibitors PgP and MRP1 have been actively pursued as targets for therapeutic suppression, since it is widely believed that safe and potent inhibitors of these
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proteins would be clinically efficacious in increasing the sensitivity of drug-resistant cancers to chemotherapy. Several MRP1-specific reversing agents have been identified in vitro and these include the leukotriene C4 antagonist MK571, the benzothipene LY329146, the dihydropiridine analogue NIK250, the quinolone antibiotic difloxacin, and the organic acids, sulfinpyrazone, benzbromarone and probenecid [4]. However, to date there are few specific clinically relevant MRP1 modulators and therefore this remains an important and ongoing area of research.
6. Conclusion The study of the role of MRP1 in neuroblastoma has provided important insights into the drug refractory behaviour of this disease, and available evidence indicates that elucidating the pathways involved in MRP1 signalling will create new approaches to improving patient outcome in this disease. Screening of small molecule chemical libraries to identify specific inhibitors of the MRP1 transporter is currently underway in our laboratory and we have identified compounds that appear to be highly effective at enhancing sensitivity to MRP1-substrate drugs both in vitro and in vivo. Such compounds may potentially provide an effective new treatment approach for neuroblastoma and other malignancies in which MRP1 plays a clinically relevant role.
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