Supplementary Figure Legends 1-3 from ODC1 Is a Critical Determinant of MYCN Oncogenesis and a Therapeutic Target in Neuroblastoma

Supplementary Figure 1 from ODC1 Is a Critical Determinant of MYCN Oncogenesis and a Therapeutic Target in Neuroblastoma

Supplementary Figure 3 from ODC1 Is a Critical Determinant of MYCN Oncogenesis and a Therapeutic Target in Neuroblastoma

Supplementary Figure 2 from ODC1 Is a Critical Determinant of MYCN Oncogenesis and a Therapeutic Target in Neuroblastoma

Embryonal cancer can arise from postnatally persistent embryonal remnant or rest cells, which are uniquely characterized by the absence of p53 mutations. Perinatal overexpression of the MycN oncoprotein in embryonal cancer precursor cells causes postnatal rests, and later tumor formation through unknown mechanisms. However, overexpression of Myc in adult tissues normally activates apoptosis and/or senescence signals as an organismal defense mechanism against cancer. Here, we show that perinatal neuroblastoma precursor cells exhibited a transiently diminished p53 response to MycN oncoprotein stress and resistance to trophic factor withdrawal, compared with their adult counterpart cells from the TH-MYCN(+/+) transgenic mouse model of neuroblastoma. The adult stem cell maintenance factor and Polycomb group protein, Bmi1 (B-cell-specific Moloney murine leukemia virus integration site), had a critical role at neuroblastoma initiation in the model, by repressing p53 responses in precursor cells. We further show in neuroblastoma tumor cells that Bmi1 could directly bind p53 in a complex with other Polycomb complex proteins, Ring1A or Ring1B, leading to increased p53 ubiquitination and degradation. Repressed p53 signal responses were also seen in precursor cells for other embryonal cancer types, medulloblastoma and acute lymphoblastic leukemia. Collectively, these date indicate a general mechanism for p53 inactivation in some embryonal cell types and consequent susceptibility to MycN oncogenesis at the point of embryonal tumor initiation.

Medulloblastoma is the most common brain tumor in children. Despite multimodal therapy, survival is poor. In this review we highlight the pathogenesis of medulloblastoma, and the signalling pathways which may contribute to medulloblastoma tumorigenesis. Medulloblastoma is a neuro-embryonal tumor that presents many cellular characteristics similar to that of the precursor cells of the embryonic cerebellum. Clarification of the cells of origin, in addition, to the mechanisms of initiation, promotion, and progression of tumor are key steps that need to be unravelled to enable the identification of new therapeutic targets for medulloblastoma.

The N-Myc oncoprotein is a critical factor in neuroblastoma tumorigenesis which requires additional mechanisms converting a low-level to a high-level N-Myc expression. N-Myc protein is stabilized when phosphorylated at Serine 62 by phosphorylated ERK protein. Here we describe a novel positive feedback loop whereby N-Myc directly induced the transcription of the class III histone deacetylase SIRT1, which in turn increased N-Myc protein stability. SIRT1 binds to Myc Box I domain of N-Myc protein to form a novel transcriptional repressor complex at gene promoter of mitogen-activated protein kinase phosphatase 3 (MKP3), leading to transcriptional repression of MKP3, ERK protein phosphorylation, N-Myc protein phosphorylation at Serine 62, and N-Myc protein stabilization. Importantly, SIRT1 was up-regulated, MKP3 down-regulated, in pre-cancerous cells, and preventative treatment with the SIRT1 inhibitor Cambinol reduced tumorigenesis in TH-MYCN transgenic mice. Our data demonstrate the important roles of SIRT1 in N-Myc oncogenesis and SIRT1 inhibitors in the prevention and therapy of N-Myc-induced neuroblastoma.

The family of tripartite-motif (TRIM) proteins are involved in diverse cellular processes, but are often characterized by critical protein–protein interactions necessary for their function. TRIM16 is induced in different cancer types, when the cancer cell is forced to proceed down a differentiation pathway. We have identified TRIM16 as a DNA-binding protein with histone acetylase activity, which is required for the retinoic acid receptor β2 transcriptional response in retinoid-treated cancer cells. In this study, we show that overexpressed TRIM16 reduced neuroblastoma cell growth, enhanced retinoid-induced differentiation and reduced tumourigenicity in vivo. TRIM16 was only expressed in the differentiated ganglion cell component of primary human neuroblastoma tumour tissues. TRIM16 bound directly to cytoplasmic vimentin and nuclear E2F1 in neuroblastoma cells. TRIM16 reduced cell motility and this required downregulation of vimentin. Retinoid treatment and enforced overexpression caused TRIM16 to translocate to the nucleus, and bind to and downregulate nuclear E2F1, required for cell replication. This study, for the first time, demonstrates that TRIM16 acts as a tumour suppressor, affecting neuritic differentiation, cell migration and replication through interactions with cytoplasmic vimentin and nuclear E2F1 in neuroblastoma cells.

Increased retinoic acid receptor β (RAR β 2 ) gene expression is a hallmark of cancer cell responsiveness to retinoid anticancer effects. Moreover, low basal or induced RAR β 2 expression is a common feature of many human cancers, suggesting that RAR β 2 may act as a tumour suppressor gene in the absence of supplemented retinoid. We have previously shown that low RAR β 2 expression is a feature of advanced neuroblastoma. Here, we demonstrate that the ABC domain of the RAR β 2 protein alone was sufficient for the growth inhibitory effects of RAR β 2 on neuroblastoma cells. ATP7A, the copper efflux pump, is a retinoid-responsive gene, was upregulated by ectopic overexpression of RAR β 2 . The ectopic overexpression of the RAR β 2 ABC domain was sufficient to induce ATP7A expression, whereas, RAR β 2 siRNA blocked the induction of ATP7A expression in retinoid-treated neuroblastoma cells. Forced downregulation of ATP7A reduced copper efflux and increased viability of retinoid-treated neuroblastoma cells. Copper supplementation enhanced cell growth and reduced retinoid-responsiveness, whereas copper chelation reduced the viability and proliferative capacity. Taken together, our data demonstrates ATP7A expression is regulated by retinoic acid receptor β and it has effects on intracellular copper levels, revealing a link between the anticancer action of retinoids and copper metabolism.

Retinoids have significant clinical activity in several human cancers, yet the factors determining retinoid sensitivity in cancer cells are still unclear. Retinoid-induced expression of retinoic acid receptor (RAR) β2 is a necessary component of the retinoid anticancer signal in cancer cells. We have previously identified the Estrogen-responsive B Box Protein (EBBP), a member of the Tripartite Motif (TRIM) protein family, as a novel RARβ2 transcriptional regulator in the retinoid signal. Here we examined the mechanism of the EBBP effect on the retinoid anticancer signal. We assessed retinoid-responsive RARβ2 transcription in retinoid-resistant breast and lung cancer cells in the presence of chromatin modifying agents. A histone deacetylase (HDAC) inhibitor alone, or in combination with retinoid, was more effective than a demethylating agent in restoring RARβ2 transcription in resistant cells. Overexpression of EBBP alone markedly increased histone acetylation. The effect of EBBP on retinoid-responsive transcription appeared to be limited to genes with the retinoic acid response element (βRARE) regulatory sequence, such as CYP26A1. EBBP inhibited cell growth by effects on cyclin D1 and Phospho-Rb, and, reduced cell viability in retinoid-resistant cancer cells. The viability of non-cancer cells was unaffected by EBBP overexpression. Taken together our data suggests that EBBP acts to de-repress transcription of RARβ2 and CYP26A1, by modifying histone acetylation in retinoid-resistant cancer cells, and, is an important target for drug discovery in retinoid-resistant cancers.

Abstract Neuroblastoma is a frequently lethal childhood tumor in which MYC gene deregulation, commonly as MYCN amplification, portends poor outcome. Identifying the requisite biopathways downstream of MYC may provide therapeutic opportunities. We used transcriptome analyses to show that MYCN-amplified neuroblastomas have coordinately deregulated myriad polyamine enzymes (including ODC1, SRM, SMS, AMD1, OAZ2, and SMOX) to enhance polyamine biosynthesis. High-risk tumors without MYCN amplification also overexpress ODC1, the rate-limiting enzyme in polyamine biosynthesis, when compared with lower-risk tumors, suggesting that this pathway may be pivotal. Indeed, elevated ODC1 (independent of MYCN amplification) was associated with reduced survival in a large independent neuroblastoma cohort. As polyamines are essential for cell survival and linked to cancer progression, we studied polyamine antagonism to test for metabolic dependence on this pathway in neuroblastoma. The Odc inhibitor α-difluoromethylornithine (DFMO) inhibited neuroblast proliferation in vitro and suppressed oncogenesis in vivo. DFMO treatment of neuroblastoma-prone genetically engineered mice (TH-MYCN) extended tumor latency and survival in homozygous mice and prevented oncogenesis in hemizygous mice. In the latter, transient Odc ablation permanently prevented tumor onset consistent with a time-limited window for embryonal tumor initiation. Importantly, we show that DFMO augments antitumor efficacy of conventional cytotoxics in vivo. This work implicates polyamine biosynthesis as an arbiter of MYCN oncogenesis and shows initial efficacy for polyamine depletion strategies in neuroblastoma, a strategy that may have utility for this and other MYC-driven embryonal tumors. [Cancer Res 2008;68(23):9735–45]

516 The majority of child cancer arises in embryonal cells which have persisted beyond birth, having failed to undergo cell death following normal organogenesis in utero. The mechanism of embryonal cell persistence, and thus, tumour initiation are unknown. Resistance to spontaneous regression by neuroblasts is an important early step in human neuroblastoma tumorigenesis. We have previously shown that perinatal expression of a MYCN transgene in paravertebral murine neuroblasts mediates transient neuroblast proliferation and resistance to apoptotic cell death, as the first step in neuroblastoma tumorigenesis (Hansford et al., PNAS 2004). Here we analysed the mechanism of resistance to apoptotic cell death in primary ganglion cells from MYCN transgenic, compared with normal mice. MYCN-mediated resistance to cell death was seen in ganglion cells following a variety of different death stimuli: nerve growth factor (NGF) withdrawal, hypoxia, nutrient deprivation and doxorubicin. The mRNA expression levels of known MycN transcriptional target genes in 2-week old primary ganglion tissue from normal and MYCN transgenic mice was then assessed. We found only induction of telomerase, α-prothymosin, multidrug resistance protein-1, MCM7 minichromosome maintenance deficient-7 and ornithine decarboxylase in MYCN mouse tissues. We next used an adenoviral gene transfer system to express deletion mutant MycN proteins in normal primary ganglion cells, and showed that only the Myc Box II domain of the full-length MycN protein was necessary for MycN-mediated resistance to NGF withdrawal. Following NGF withdrawal, ganglion cells from MYCN mice demonstrated markedly reduced nuclear translocation of both apoptosis-inducing factor and caspase-activated DNAse. The response of regulatory and effector proteins in the known apoptosis pathway activated by NGF withdrawal was then studied. Following NGF withdrawal, MYCN transgenic and normal ganglion cells did not differ for the phosphorylation responses of Jun kinase, ATF2, or c-Jun, or nuclear translocation of FOXO3a and consequent Bim transcription. In contrast, translocation of Bax to mitochondria was significantly reduced in MYCN, compared with normal, ganglion cells. Taken together our data indicate a MycN-driven mechanism for death resistance in neuroblasts involving the Myc Box II domain of the MycN protein and Bax translocation, which may represent a general mechanism for embryonal cancer initiation.

Retinoic acid (RA) induces growth arrest, cell death, and differentiation in many human cancer cells in vitro and has entered routine clinical use for the treatment of several human cancer types. One mechanism by which cancer cells evade retinoid-induced effects is through repression of retinoic acid receptor beta (RAR beta) gene transcription. The RA response element beta (beta RARE) is the essential DNA sequence required for retinoid-induced RAR beta transcription. Here we show that the estrogen-responsive B box protein (EBBP), a member of the RING-B box-coiled-coil protein family, is a beta RARE-binding protein. EBBP undergoes serine threonine phosphorylation and enhanced protein stability after RA treatment. Following RA treatment, we also observed increased nuclear EBBP levels in aggregates with the promyelocytic leukemia protein at promyelocytic leukemia nuclear bodies. EBBP enhanced RA-responsive RAR beta transcription in RA-sensitive and -resistant cancer cells, which were resistant to both a histone deacetylase inhibitor and a demethylating agent. EBBP-specific small interfering RNA reduced basal and RA-induced RAR beta expression. EBBP increased beta RARE-transactivating function through its coiled-coil domain. Taken together, our work suggests that EBBP may have a pivotal role in the retinoid anti-cancer signal.

N-myc has emerged as a member of a transcriptional regulatory network which impinges directly on the machinery of cell growth and proliferation. Critical during neural crest embryogenesis, N-myc is rapidly down-regulated as tissues become terminally differentiated and growth-arrested. The involvement of N-myc in these fundamental cellular processes necessitates an intricate strategy for its regulation, which is still being elucidated. Deregulated N-myc over-expression has clear transforming ability in vitro and in vivo. The transcriptional target genes responsible for this activity are beginning to be unravelled.

The mechanisms causing persistence of embryonal cells that later give rise to tumors is unknown. One tumorigenic factor in the embryonal childhood tumor neuroblastoma is the MYCN protooncogene. Here we show that normal mice developed neuroblast hyperplasia in paravertebral ganglia at birth that completely regressed by 2 weeks of age. In contrast, ganglia from MYCN transgenic (TH-MYCN) mice demonstrated a marked increase in neuroblast hyperplasia and MycN expression during week 1. Regression of neuroblast hyperplasia was then delayed and incomplete before neuroblastoma tumor formation at 6 and 13 weeks in homo- and hemizygote mice, respectively. Paravertebral neuronal cells cultured from perinatal TH-MYCN mice exhibited 3- to 10-fold resistance to nerve growth factor (NGF) withdrawal, compared with normal mice. Both low- and high-affinity NGF receptors were expressed in perinatal neuroblast hyperplasia but not in neuroblastoma tumor tissue. MYCN transgene amplification was present at low levels in perinatal neuroblast hyperplasia from both homo-and hemizygote TH-MYCN mice. However, only in hemizygous mice did tumor formation correlate with a stepwise increase in the frequency of MYCN amplification. These data suggest that inappropriate perinatal MycN expression in paravertebral ganglia cells from TH-MYCN mice initiated tumorigenesis by altering the physiologic process of neural crest cell deletion. Persisting embryonal neural crest cells underwent further changes, such as MYCN amplification and repression of NGF receptor expression, during tumor progression. Our studies provide a model for studying perinatal factors influencing embryonal tumor initiation.

3838 Despite recent advances in the treatment of high-risk category neuroblastoma (NB) patient with 13-cis retinoic acid therapy, the survival rate of these patients remains low, at less than 30%, due to the emergence multi-drug resistant minimal residual NB cells. Resistance to chemo and retinoid therapy in NB has been linked to a number of factors including high expression and amplification of the poor prognostic marker mycN gene, high level of Bcl-2, non-functional p53 and p21. Histone deacetylase (HDAC) inhibitors have shown remarkable anti-cancer activities against several types of cancer. In this study, we aim to evaluate the therapeutic efficacy of hydroxamic acid-based HDAC inhibitor trichostatin A (TSA) in the mycN transgenic mouse model of human NB and its ability to enhance the efficacy of retinoid and interferons (IFNs) therapy, and in addition, to identify factors that may mediate resistance to HDAC inhibitor. TSA reduces NB tumor growth by over 50% in mycN transgenic mice at dosages that have no clinically observable toxicity. Furthermore, our data showed that resistance to TSA-induced apoptosis correlates with high level of intrinsic expression of p21 protein but is independent of expression level of Bcl-2, p53 status, mycN gene expression or amplification. Multi-drug resistance protein (MRP) phenotypes do not increase resistance to TSA-induced apoptosis in NB cell lines. TSA, at a lower concentration, significantly enhances the anti-cancer effect of retinoid against NB cell lines; however, the combination of TSA and IFN-alpha showed the strongest effect and synergy. Taken together, our pre-clinical data showed that hydroxamic acid-based HDAC inhibitor has high therapeutic efficacy and selectivity against aggressive NB both in vitro and in vivo models. Furthermore, combining HDAC inhibitor therapy with IFN-alpha may significantly increase the therapeutic benefit of this new class of anti-cancer agent. These findings provides a rational basis for the inclusion of HDAC inhibitor and IFN-alpha as part of the treatment for high-risk NB patients.