MDM2 as MYCN transcriptional target: Implications for neuroblastoma pathogenesis

Cancer Letters 228 (2005) 21–27 www.elsevier.com/locate/canlet

MDM2 as MYCN transcriptional target: Implications for neuroblastoma pathogenesis Andrew Slacka, Guillermina Lozanob, Jason M. Shoheta,* a

Department of Pediatrics, Texas Children’s Cancer Center and Center for Cell and Gene Therapy, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA b Department of Molecular Genetics, Section of Cancer Genetics, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030, USA Received 15 December 2004; accepted 12 January 2005

Abstract MYCN amplification is associated with an exceptionally poor prognosis in neuroblastoma. Furthermore, the crucial effectors of MYCN responsible for this aggressive subset of neuroblastoma await characterization. A critical negative regulator of the p53 tumor suppressor, MDM2, has been recently characterized in neuroblastoma cell lines as a transcriptional target of MYCN. Targeted inhibition of MYCN results in reduced MDM2 expression levels, with concomitant stabilization of p53 and stimulation of apoptosis in MYCN amplified neuroblastoma cell lines. These data suggest the possibility that MYCN-driven expression of MDM2 might play a role in counterbalancing the p53-dependent apoptotic pathways concurrently stimulated by over expression of MYC proteins. Mouse models of lymphoma have demonstrated that MDM2 expression, with decreased p53 activity, is critical for complete MYCC driven tumorigenesis. Our data suggest that a similar situation may apply for MYCN in neuroblastoma. Strategies for pharmacologic and genetic inhibition of MDM2 may prove to be an important new therapeutic approach in neuroblastoma. q 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: MYCN; MDM2; Apoptosis; Neuroblastoma; p53

1. Introduction and background Major improvements in outcome for neuroblastoma are most likely to result from targeted molecular therapies, derived from an advanced understanding of the molecular pathogenesis of the disease. MYCN amplification is the strongest adverse prognostic * Corresponding author. Tel.: C31 20 5666592. E-mail address: [email protected] (J.M. Shohet).

factor in neuroblastoma treatment [1] and transgenic models of neuroblastoma have revealed that MYCN is also a transforming oncogene responsible for de novo tumor formation [2]. MYCN is involved in many aspects of normal and oncogenic cellular physiology via activation of various transcriptional targets. These target genes encode proteins with roles in proliferation, cell cycle regulation, apoptosis and genomic stability [3,4]. In vitro studies demonstrate that MYCN overexpression induces an aggressive metastatic phenotype with decreased contact inhibition,

0304-3835/$ - see front matter q 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2005.01.050

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decreased growth factor dependence and increased proliferation rate [5–7]. Paradoxically, MYCN also suppresses Bcl-2, activates Bax, and sensitizes cells to genotoxicitymediated apoptosis through intrinsic apoptosis pathways [8]. MYCC (a MYC family member) has also been shown to activate the ARF tumor suppressor leading to p53 activation and apoptosis through Bcl-x and Bcl-2-dependent and independent pathways [9]. Thus MYCN expression induces the apparently contradictory cellular processes of rapid proliferation and apoptotic cell death. Obligate defects in the apoptotic pathways accompanying MYCN amplification have been proposed to circumvent MYCNstimulated cell death in neuroblastoma [10]. Characterization of the key downstream transcriptional targets of MYCN involved in these conflicting pathways will yield important insight into the pathogenesis of and novel therapeutic approaches to neuroblastoma.

2. MDM2 is a MYCN target Using chromatin immunoprecipitation (ChIP) cloning [11], we recently characterized MDM2, the essential negative regulator of the p53 tumor suppressor, as a transcriptional target of the MYCN oncogene in neuroblastoma [12]. Real time PCR, Western blots and luciferase reporter assays demonstrate MYCN dependent regulation of MDM2 in MYCN-inducible neuroblastoma cell lines. ChIP and promoter pull-down assays demonstrate that this transcriptional regulation was through a direct interaction with a canonical E-box in the MDM2 promoter. This study also shows that targeted inhibition of MYCN leads to decreased MDM2 and consequently increased p53 mediated apoptosis. In contrast to many tumors, less than 2% of neuroblastomas have mutated p53 and the p53 pathways are functionally active in the majority of de novo tumors [13,14]. The characterization of MDM2 as a direct transcriptional target of MYCN suggests a mechanism by which MYCN overexpression might destabilize p53, thereby raising the threshold for stimulation of growth arrest and apoptosis. As detailed below, inhibition of the MDM2/ p53 pathway in vivo may tip the balance between

MYCN driven proliferation and apoptosis in favor of apoptosis through activation of p53.

3. MDM2 expression as a critical determinant in cancer Originally identified as an amplified gene located on a double minute chromosome in a transformed mouse cell line [15], Mdm2 was later shown to have a critical role in the process of cellular transformation [16]. Amplification and overexpression of MDM2 is found in about 10% of all human tumors [17]. In gliomas, for example, MDM2 amplification identifies a subset of high-risk patients that do not have p53 mutations [18]. In many soft tissue sarcomas, MDM2 amplification and overexpression correlates with poor prognosis [19–21]. Studies of acute lymphoblastic leukemia and non-Hodgkin’s lymphoma also demonstrate that MDM2 over expression is associated with poor survival and aggressive disease [22–24]. Validating animal studies (described below) demonstrate that Mdm2 gene dosage is critical for oncogene driven lymphomagenesis. Thus deregulation of MDM2 gene expression likely contributes to the pathogenesis of a wide range of human tumors.

4. MDM2 functions in normal and malignant cells Upon genotoxic or cellular stress, increased p53 activity transactivates numerous genes encoding effectors critical in apoptosis and cell cycle arrest [25]. Additionally, p53 activates the MDM2 gene that encodes a p53 inhibitor forming an autoregulatory feedback loop that tightly controls expression of both p53 and MDM2 [26]. Multiple MDM2 activities inhibit p53 function. MDM2 binds to p53 via an Nterminal domain and directly inhibits the ability of that protein to transactivate its target genes [27]. MDM2 also directs the export of p53 away from its site of action in the nucleus to the cytoplasm, thanks to a central nuclear export signal [28,29]. Finally, MDM2 helps to target p53 for degradation through its E3 ubiquitin ligase activity [30–32]. MDM2 also possesses p53 independent, cell-cycle specific functions relevant to tumorigenesis. These include regulation of p21WAF1/CIP1 protein

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stability [33], stimulation of E2F1/DP1 transcriptional activity [34], inhibition of RB function in the regulation of E2F1 [35], and blockade of p300/CBP acetylation of p53 [36]. Mouse models demonstrate a role for Mdm2 in developmental regulation and differentiation [37]. Overall, MDM2 permits proliferation through inhibition of p53-mediated arrest and apoptosis, as well through multiple p53-independent mechanisms pertinent to the process of tumorigenesis (see [17] for review).

5. MDM2 functions in neuroblastoma Less than 2% of de novo neuroblastomas harbor mutations in p53 [38]. Recent literature demonstrates that p53 is generally functional, accumulates in the nucleus in response to DNA damage and is efficiently degraded by MDM2 in neuroblastoma [39–41]. There are reports that p53 function may be compromised as a consequence of aberrant MDM2 expression levels in neuroblastoma. For instance, a study examining etoposide-induced p53 activity in a neuroblastoma cell lines suggested an important role for elevated MDM2 expression levels in the regulation of p53 translocation. In SH-EP cells, p53 accumulates in the cytoplasm upon exposure to etoposide. Antisense inhibition of MDM2 can then restore predicted nuclear localization and apoptotic activation, suggesting that increased MDM2 expression levels can play a role in the inappropriate modulation of p53 function [42]. Additionally, elevated MDM2 expression, and accompanying loss of p53 function is associated with multidrug resistance in some neuroblastoma cell lines [43]. The activity of MDM2 is critical in neuroblastoma, as recent work demonstrates that MDM2 ubiquitin ligase activity is rate-limiting for p53 degradation in neuroblastoma [44]. Taken together, these data demonstrate that the MDM2/p53 pathway is intact in neuroblastoma and suggest that deficiencies in p53 functions may be a consequence of aberrant MDM2 expression. Deregulation of MDM2 as an effector of MYCN could therefore significantly diminish the activity of p53 during neuroblastoma development, resulting in failure to undergo appropriate cell cycle arrest and apoptosis (see below).

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6. Transgenic mouse models demonstrate a role for MDM2 in cancer The importance of MDM2 as a negative regulator of p53 is exemplified in a mouse model. Loss of Mdm2 in mice leads to cell death (apoptosis) at the blastocyst stage and embryonic lethality [45–47]. This phenotype is completely p53-dependent as mice lacking both Mdm2 and p53 are viable [45,46]. As adults, Mdm2/p53 double null mice have the same tumor phenotype as p53 null mice [48]. Thus, the most important in vivo function of Mdm2 seems to be to inhibit p53 activity, which is detrimental to cell viability. The availability of an Mdm2 null allele allowed a direct assessment of the importance of mdm2 in tumor development in another mouse model. Mice expressing the Em-myc transgene develop B- cell lymphomas and as such, represent a model for Burkitt’s lymphoma [49]. Mdm2 haploinsufficiency in Em-myc mice profoundly delayed tumorigenesis [50]. Mice expressing Em-myc develop B-cell lymphomas with a median age of 20.6 weeks while mice expressing Em-myc and inheriting an Mdm2 null allele developed tumors much later with a median age of 44.3 weeks. These data suggest that decreased levels of MDM2 lead to increased p53 activity which delays susceptibility to Em-myc lymphomas. Indeed, the increased apoptosis in Em-myc Mdm2 C/K pre-B cells is completely rescued by deletion of p53. The ability of MDM2 levels to alter tumor susceptibility has also been observed in human cancers. A polymorphism in the human MDM2 promoter was recently identified that modulates expression of MDM2 via binding of the transcription factor Sp1 [51]. This polymorphism results in increased MDM2 levels with a concomitant attenuation of p53 levels, and its presence correlates with increased susceptibility to tumor incidence in hereditary as well as sporadic cancers. Thus, minor differences in the expression of MDM2 alter tumorigenesis in animal and human cancers.

7. MDM2 as an effector of the MYCN oncogene A longstanding paradox regarding the MYC family of oncogenes is their propensity to activate apoptotic

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Fig. 1. MYC dependent apoptotic, growth arrest and proliferative and pathways. MYCN-mediated apoptotic and cell cycle arrest pathways are indicated by light arrows. Heavy arrows indicate MYCN-mediated pathways promoting proliferation via MDM2. MYC activates ARF and inhibits Bcl family members leading to apoptosis. MDM2 counteracts these influences by inhibiting p53 leading to repressed cell cycle arrest/apoptosis and promoting cell cycle progression and proliferation.

pathways stimulating cell death and to simultaneously promote cell cycle progression. Induction of MYCN in neuroblastoma cell lines, for instance, promotes enhanced sensitivity to apoptotic stimuli [52], yet stimulates accelerated G1-S progression [5]. Deficiencies in various components of apoptotic pathways, including caspase 8 inactivation and enhanced Bcl-2 and survivin expression levels accompany MYCN amplification in neuroblastoma [53–56] and ensure continued proliferation. We now propose a model whereby MYCN transactivation of MDM2 may also, in part, be responsible for offsetting the multiple apoptotic pathways stimulated by MYC. As shown in Fig. 1, MYC promotes apoptosis by inhibiting functions of Bcl-2/Bcl-XL, which protect the cell from the proapototic activity of Bax [57]. MYC also enhances expression of the p19ARF (ARF) tumor suppressor [58] that sequesters MDM2 in the nucleolus [59,60]. This prevents MDM2-p53 binding and shuttling of p53 from the nucleus for degradation, thereby promoting apoptosis and growth arrest stimulated by p53. Our data supports the hypothesis that MDM2 overexpression in response to MYCN shortens the p53 half-life and/or raises the threshold for activation

of p53 by cellular stress, counteracting the opposing effects MYCN on ARF and Bcl-2/BclxL pathways. This novel molecular link between MYCN and MDM2/p53 pathway suggests an important new layer of regulation promoting growth and proliferation in response to MYCN amplification. Additional evidence implicating MYCN driven aberrant gene expression in the pathogenesis of neuroblastoma comes from the recent work describing hyperproliferation of the sympathetic ganglia of the PTH-MYCN neuroblastoma mouse model [2]. These mice have MYCN expression targeted to neural crest derived tissues under the control of the tyrosine hydroxylase promoter [2]. Ganglia isolated from homozygous mice exhibit persistence of hyperproliferative islands of cells thought to be pre-malignant neuroblasts. The ganglia neuroblasts undergo rapid differentiation with high apoptosis rates under the direction of local trophic factors. Persistence of the hyperproliferative clusters beyond 1wk of age correlates with high rates of neuroblastoma development [61]. It is likely that MYCN is disrupting the normal differentiation response in these cells. MYCN driven elevation in MDM2 levels could well contribute to this phenomenon since high MDM2 levels should alter the cellular response to stress, promoting cell cycle progression at this critical stage of neuroblast development.

8. Implications for novel therapies The biological characteristics of neuroblastoma suggest that targeting the molecular interaction of MDM2 and p53 may be an effective therapeutic strategy. As noted, the majority of neuroblastomas are p53 wild type and therefore dependent on MDM2 regulation of p53 to prevent apoptotic cell death. In several other tumor models inhibition of MDM2 leads to increased p53 activity and triggers an apoptotic stress response [62–64]. In neuroblastoma cell lines, we recently demonstrated that down regulation of MYCN leads to decreased MDM2 expression, p53 stabilization and subsequent rapid cell death [12]. Since MYCN also sensitizes cells to apoptotic stress, neuroblastoma should be particularly sensitive to targeted disruption of the MDM2/p53 interaction in vivo.

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MDM2 as a drug target is the topic of extensive reviews and is an active area of research. Small molecule inhibitors that specifically interfere with the binding of MDM2 to p53 have been shown to induce apoptosis and regression of osteosarcoma xenografts [62]. The unique developmental biology of neuroblastoma suggests to us that targeting MDM2 could be effective therapy, especially in the context of MYCN driven tumors. The future evaluation of such approaches will advance our understanding of neuroblastoma pathogenesis and hopefully lead to important clinical advances for this highly fatal pediatric malignancy.

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