Eric Sekyere

ODC1 Is a Critical Determinant of MYCN Oncogenesis and a Therapeutic Target in Neuroblastoma

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 alpha-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.

SIRT1 Promotes N-Myc Oncogenesis through a Positive Feedback Loop Involving the Effects of MKP3 and ERK on N-Myc Protein Stability

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.

Activation of tissue transglutaminase transcription by histone deacetylase inhibition as a therapeutic approach for Myc oncogenesis

Histone deacetylase (HDAC) inhibitors reactivate tumor suppressor gene transcription; induce cancer cell differentiation, growth arrest, and programmed cell death; and are among the most promising new classes of anticancer drugs. Myc oncoproteins can block cell differentiation and promote cell proliferation and malignant transformation, in some cases by modulating target gene transcription. Here, we show that tissue transglutaminase (TG2) was commonly reactivated by HDAC inhibitors in neuroblastoma and breast cancer cells but not normal cells and contributed to HDAC inhibitor-induced growth arrest. TG2 was the gene most significantly repressed by N-Myc in neuroblastoma cells in a cDNA microarray analysis and was commonly repressed by N-Myc in neuroblastoma cells and c-Myc in breast cancer cells. Repression of TG2 expression by N-Myc in neuroblastoma cells was necessary for the inhibitory effect of N-Myc on neuroblastoma cell differentiation. Dual step cross-linking chromatin immunoprecipitation and protein coimmunoprecipitation assays showed that N-Myc acted as a transrepressor by recruiting the HDAC1 protein to an Sp1-binding site in the TG2 core promoter in a manner distinct from its action as a transactivator at E-Box binding sites. HDAC inhibitor treatment blocked the N-Myc-mediated HDAC1 recruitment and TG2 repression in vitro. In neuroblastoma-bearing N-Myc transgenic mice, HDAC inhibitor treatment induced TG2 expression and demonstrated marked antitumor activity in vivo. Taken together, our data indicate the critical roles of HDAC1 and TG2 in Myc-induced oncogenesis and have significant implications for the use of HDAC inhibitor therapy in Myc-driven oncogenesis.

Transcriptional upregulation of histone deacetylase 2 promotes Myc-induced oncogenic effects

Myc oncoproteins and histone deacetylases (HDACs) modulate gene transcription and enhance cancer cell proliferation, and HDAC inhibitors are among the most promising new classes of anticancer drugs. Here, we show that N-Myc and c-Myc upregulated HDAC2 gene expression in neuroblastoma and pancreatic cancer cells, respectively, which contributed to N-Myc- and c-Myc-induced cell proliferation. Cyclin G2 (CCNG2) was commonly repressed by N-Myc and HDAC2 in neuroblastoma cells and by c-Myc and HDAC2 in pancreatic cancer cells, and could be reactivated by HDAC inhibitors. 5-bromo-2′-deoxyuridine incorporation assays showed that transcriptional repression of CCNG2 was, in part, responsible for N-Myc-, c-Myc- and HDAC2-induced cell proliferation. Dual crosslinking chromatin immunoprecipitation assay demonstrated that N-Myc acted as a transrepressor by recruiting the HDAC2 protein to Sp1-binding sites at the CCNG2 gene core promoter. Moreover, HDAC2 was upregulated, and CCNG2 downregulated, in pre-cancerous and neuroblastoma tissues from N-Myc transgenic mice, and c-Myc overexpression correlated with upregulation of HDAC2 and repression of CCNG2 in tumour tissues from pancreatic cancer patients. Taken together, our data indicate the critical roles of upregulation of HDAC2 and suppression of CCNG2 in Myc-induced oncogenesis, and have significant implications for the application of HDAC inhibitors in the prevention and treatment of Myc-driven cancers.

The membrane-bound transferrin homologue melanotransferrin: roles other than iron transport?

Melanotransferrin (MTf) is a membrane‐bound transferrin (Tf) homologue that is found at high levels in melanoma cells. MTf has many characteristics in common with serum Tf and previous studies have shown that it can bind Fe. This has led to speculation that MTf may be involved in Fe transport. Because Fe is required for a variety of metabolic reactions including ATP and DNA synthesis, MTf could play a role in proliferation. However, recently it has been shown that MTf plays very little role in Fe uptake by melanoma cells, and unlike other Fe transport molecules (e.g. the transferrin receptor), its expression is not controlled by Fe. In the present review the function of MTf is discussed in relation to data suggesting other roles apart from Fe uptake.

Direct effects of Bmi1 on p53 protein stability inactivates oncoprotein stress responses in embryonal cancer precursor cells at tumor initiation

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.

Patched1 deletion increases N-Myc protein stability as a mechanism of medulloblastoma initiation and progression

Medulloblastoma tumorigenesis caused by inactivating mutations in the PATCHED1 (PTCH1) gene is initiated by persistently activated Sonic Hedgehog (Shh) signaling in granule neuron precursors (GNPs) during the late stages of cerebellar development. Both normal cerebellar development and Shh-driven medulloblastoma tumorigenesis require N-Myc expression. However, the mechanisms by which N-Myc affects the stages of medulloblastoma initiation and progression are unknown. Here we used a mouse model of Ptch1 heterozygosity and medulloblastoma to show that increased N-Myc expression characterized the earliest selection of focal GNP hyperplasia destined for later tumor progression. Step-wise loss of Ptch1 expression, from tumor initiation to progression, led to incremental increases in N-Myc protein, rather than mRNA, expression. Increased N-Myc resulted in enhanced proliferation and death resistance of perinatal GNPs at tumor initiation. Sequential N-Myc protein phosphorylation at serine-62 and serine-62/threonine-58 characterized the early and late stages of medulloblastoma tumorigenesis, respectively. Shh pathway activation led to increased Myc protein stability and reduced expression of key regulatory factors. Taken together our data identify N-Myc protein stability as the result of loss of Ptch1, which distinguishes normal cerebellar development from medulloblastoma tumorigenesis.

TRIM16 acts as a tumour suppressor by inhibitory effects on cytoplasmic vimentin and nuclear E2F1 in neuroblastoma cells

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.

Serial Transcriptome Analysis and Cross-Species Integration Identifies Centromere-Associated Protein E as a Novel Neuroblastoma Target

Cancer genomic studies that rely on analysis of biopsies from primary tumors may not fully identify the molecular events associated with tumor progression. We hypothesized that characterizing the transcriptome during tumor progression in the TH-MYCN transgenic model would identify oncogenic drivers that would be targetable therapeutically. We quantified expression of 32,381 murine genes in nine hyperplastic ganglia harvested at three time points and four tumor cohorts of progressively larger size in mice homozygous for the TH-MYCN transgene. We found 93 genes that showed a linearly increasing or decreasing pattern of expression from the preneoplastic ganglia to end stage tumors. Cross-species integration identified 24 genes that were highly expressed in human MYCN-amplified neuroblastomas. The genes prioritized were not exclusively driven by increasing Myc transactivation or proliferative rate. We prioritized three targets [centromere-associated protein E (Cenpe), Gpr49, and inosine monophosphate dehydrogenase type II] with previously determined roles in cancer. Using siRNA knockdown in human neuroblastoma cell lines, we further prioritized CENPE due to inhibition of cellular proliferation. Targeting CENPE with the small molecular inhibitor GSK923295 showed inhibition of in vitro proliferation of 19 neuroblastoma cell lines (median IC(50), 41 nmol/L; range, 27-266 nmol/L) and delayed tumor growth in three xenograft models (P values ranged from P < 0.0001 to P = 0.018). We provide preclinical validation that serial transcriptome analysis of a transgenic mouse model followed by cross-species integration is a useful method to identify therapeutic targets and identify CENPE as a novel therapeutic candidate in neuroblastoma.

The retinoid signalling molecule, TRIM16, is repressed during squamous cell carcinoma skin carcinogenesis in vivo and reduces skin cancer cell migration in vitro

Retinoid therapy is used for chemo‐prevention in immuno‐suppressed patients at high risk of developing skin cancer. The retinoid signalling molecule, tripartite motif protein 16 (TRIM16), is a regulator of keratinocyte differentiation and a tumour suppressor in retinoid‐sensitive neuroblastoma. We sought to determine the role of TRIM16 in skin squamous cell carcinoma (SCC) pathogenesis. We have shown that TRIM16 expression was markedly reduced during the histological progression from normal skin to actinic keratosis and SCC. SCC cell lines exhibited lower cytoplasmic and nuclear TRIM16 expression compared with primary human keratinocyte (PHK) cells due to reduced TRIM16 protein stability. Overexpressed TRIM16 translocated to the nucleus, inducing growth arrest and cell differentiation. In SCC cells, TRIM16 bound to and down regulated nuclear E2F1, this is required for cell replication. Retinoid treatment increased nuclear TRIM16 expression in retinoid‐sensitive PHK cells, but not in retinoid‐resistant SCC cells. Overexpression of TRIM16 reduced SCC cell migration, which required the C‐terminal RET finger protein (RFP)‐like domain of TRIM16. The mesenchymal intermediate filament protein, vimentin, was directly bound and down‐regulated by TRIM16 and was required for TRIM16‐reduced cell migration. Taken together, our data suggest that loss of TRIM16 expression plays an important role in the development of cutaneous SCC and is a determinant of retinoid sensitivity. Copyright