Samuele Gherardi

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.

ABCC Multidrug Transporters in Childhood Neuroblastoma: Clinical and Biological Effects Independent of Cytotoxic Drug Efflux

Background Although the prognostic value of the ATP-binding cassette, subfamily C (ABCC) transporters in childhood neuroblastoma is usually attributed to their role in cytotoxic drug efflux, certain observations have suggested that these multidrug transporters might contribute to the malignant phenotype independent of cytotoxic drug efflux. Methods A v-myc myelocytomatosis viral related oncogene, neuroblastoma derived (MYCN)–driven transgenic mouse neuroblastoma model was crossed with an Abcc1-deficient mouse strain (658 hMYCN1/−, 205 hMYCN+/1 mice) or, alternatively, treated with the ABCC1 inhibitor, Reversan (n = 20). ABCC genes were suppressed using short interfering RNA or overexpressed by stable transfection in neuroblastoma cell lines BE(2)-C, SH-EP, and SH-SY5Y, which were then assessed for wound closure ability, clonogenic capacity, morphological differentiation, and cell growth. Real-time quantitative polymerase chain reaction was used to examine the clinical significance of ABCC family gene expression in a large prospectively accrued cohort of patients (n = 209) with primary neuroblastomas. Kaplan–Meier survival analysis and Cox regression were used to test for associations with event-free and overall survival. Except where noted, all statistical tests were two-sided. Results Inhibition of ABCC1 statistically significantly inhibited neuroblastoma development in hMYCN transgenic mice (mean age for palpable tumor: treated mice, 47.2 days; control mice, 41.9 days; hazard ratio [HR] = 9.3, 95% confidence interval [CI] = 2.65 to 32; P < .001). Suppression of ABCC1 in vitro inhibited wound closure (P < .001) and clonogenicity (P = .006); suppression of ABCC4 enhanced morphological differentiation (P < .001) and inhibited cell growth (P < .001). Analysis of 209 neuroblastoma patient tumors revealed that, in contrast with ABCC1 and ABCC4, low rather than high ABCC3 expression was associated with reduced event-free survival (HR of recurrence or death = 2.4, 95% CI = 1.4 to 4.2; P = .001), with 23 of 53 patients with low ABCC3 expression experiencing recurrence or death compared with 31 of 155 patients with high ABCC3. Moreover, overexpression of ABCC3 in vitro inhibited neuroblastoma cell migration (P < .001) and clonogenicity (P = .03). The combined expression of ABCC1, ABCC3, and ABCC4 was associated with patients having an adverse event, such that of the 12 patients with the “poor prognosis” expression pattern, 10 experienced recurrence or death (HR of recurrence or death = 12.3, 95% CI = 6 to 27; P < .001). Conclusion ABCC transporters can affect neuroblastoma biology independently of their role in chemotherapeutic drug efflux, enhancing their potential as targets for therapeutic intervention.

p53 Is a Direct Transcriptional Target of MYCN in Neuroblastoma

MYCN amplification occurs in approximately 25% of neuroblastomas, where it is associated with rapid tumor progression and poor prognosis. MYCN plays a paradoxical role in driving cellular proliferation and inducing apoptosis. Based on observations of nuclear p53 accumulation in neuroblastoma, we hypothesized that MYCN may regulate p53 in this setting. Immunohistochemical analysis of 82 neuroblastoma tumors showed an association of high p53 expression with MYCN expression and amplification. In a panel of 5 MYCN-amplified and 5 nonamplified neuroblastoma cell lines, and also in the Tet21N-regulatable MYCN expression system, we further documented a correlation between the expression of MYCN and p53. In MYCN-amplified neuroblastoma cell lines, MYCN knockdown decreased p53 expression. In Tet21N MYCN+ cells, higher levels of p53 transcription, mRNA, and protein were observed relative to Tet21N MYCN- cells. In chromatin immunoprecipitation and reporter gene assays, MYCN bound directly to a Myc E-Box DNA binding motif located close to the transcriptional start site within the p53 promoter, where it could initiate transcription. E-Box mutation decreased MYCN-driven transcriptional activation. Microarray analysis of Tet21N MYCN+/- cells identified several p53-regulated genes that were upregulated in the presence of MYCN, including MDM2 and PUMA, the levels of which were reduced by MYCN knockdown. We concluded that MYCN transcriptionally upregulates p53 in neuroblastoma and uses p53 to mediate a key mechanism of apoptosis.

A SP1/MIZ1/MYCN Repression Complex Recruits HDAC1 at the TRKA and p75NTR Promoters and Affects Neuroblastoma Malignancy by Inhibiting the Cell Response to NGF

Neuroblastoma is the most common extracranial solid tumor of childhood. One important factor that predicts a favorable prognosis is the robust expression of the TRKA and p75NTR neurotrophin receptor genes. Interestingly, TRKA and p75NTR expression is often attenuated in aggressive MYCN-amplified tumors, suggesting a causal link between elevated MYCN activity and the transcriptional repression of TRKA and p75NTR, but the precise mechanisms involved are unclear. Here, we show that MYCN acts directly to repress TRKA and p75NTR gene transcription. Specifically, we found that MYCN levels were critical for repression and that MYCN targeted proximal/core promoter regions by forming a repression complex with transcription factors SP1 and MIZ1. When bound to the TRKA and p75NTR promoters, MYCN recruited the histone deacetylase HDAC1 to induce a repressed chromatin state. Forced re-expression of endogenous TRKA and p75NTR with exposure to the HDAC inhibitor TSA sensitized neuroblastoma cells to NGF-mediated apoptosis. By directly connecting MYCN to the repression of TRKA and p75NTR, our findings establish a key pathway of clinical pathogenicity and aggressiveness in neuroblastoma.

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.

Direct and Coordinate Regulation of ATP-binding Cassette Transporter Genes by Myc Factors Generates Specific Transcription Signatures That Significantly Affect the Chemoresistance Phenotype of Cancer Cells

Increased expression of specific ATP-binding cassette (ABC) transporters is known to mediate the efflux of chemotherapeutic agents from cancer cells. Therefore, establishing how ABC transporter genes are controlled at their transcription level may help provide insight into the role of these multifaceted transporters in the malignant phenotype. We have investigated ABC transporter gene expression in a large neuroblastoma data set of 251 tumor samples. Clustering analysis demonstrated a strong association between differential ABC gene expression patterns in tumor samples and amplification of the MYCN oncogene, suggesting a correlation with MYCN function. Using expression profiling and chromatin immunoprecipitation studies, we show that MYCN oncoprotein coordinately regulates transcription of specific ABC transporter genes, by acting as either an activator or a repressor. Finally, we extend these notions to c-MYC showing that it can also regulate the same set of ABC transporter genes in other tumor cells through similar dynamics. Overall our findings provide insight into MYC-driven molecular mechanisms that contribute to coordinate transcriptional regulation of a large set of ABC transporter genes, thus affecting global drug efflux.

Physical interaction between MYCN oncogene and polycomb repressive complex 2 (PRC2) in neuroblastoma: functional and therapeutic implications.

Background: The neuroblastoma oncogene MYCN and the PRC2 members EZH2 and SUZ12 are regulators of gene transcription. Results: MYCN and PRC2 form a repressive complex on the promoter of the tumor suppressor gene CLU. Conclusion: PRC2 members are recruited by MYCN to repress gene expression and induce tumorigenesis. Significance: Reactivation of MYCN-PRC2-repressed genes by epigenetic drugs could be of clinical value in neuroblastoma. CLU (clusterin) is a tumor suppressor gene that we have previously shown to be negatively modulated by the MYCN proto-oncogene, but the mechanism of repression was unclear. Here, we show that MYCN inhibits the expression of CLU by direct interaction with the non-canonical E box sequence CACGCG in the 5′-flanking region. Binding of MYCN to the CLU gene induces bivalent epigenetic marks and recruitment of repressive proteins such as histone deacetylases and Polycomb members. MYCN physically binds in vitro and in vivo to EZH2, a component of the Polycomb repressive complex 2, required to repress CLU. Notably, EZH2 interacts with the Myc box domain 3, a segment of MYC known to be essential for its transforming effects. The expression of CLU can be restored in MYCN-amplified cells by epigenetic drugs with therapeutic results. Importantly, the anticancer effects of the drugs are ablated if CLU expression is blunted by RNA interference. Our study implies that MYC tumorigenesis can be effectively antagonized by epigenetic drugs that interfere with the recruitment of chromatin modifiers at repressive E boxes of tumor suppressor genes such as CLU.

The DMD Locus Harbours Multiple Long Non-Coding RNAs Which Orchestrate and Control Transcription of Muscle Dystrophin mRNA Isoforms.

The 2.2 Mb long dystrophin (DMD) gene, the largest gene in the human genome, corresponds to roughly 0.1% of the entire human DNA sequence. Mutations in this gene cause Duchenne muscular dystrophy and other milder X-linked, recessive dystrophinopathies. Using a custom-made tiling array, specifically designed for the DMD locus, we identified a variety of novel long non-coding RNAs (lncRNAs), both sense and antisense oriented, whose expression profiles mirror that of DMD gene. Importantly, these transcripts are intronic in origin and specifically localized to the nucleus and are transcribed contextually with dystrophin isoforms or primed by MyoD-induced myogenic differentiation. Furthermore, their forced ectopic expression in both human muscle and neuronal cells causes a specific and negative regulation of endogenous dystrophin full length isoforms and significantly down-regulate the activity of a luciferase reporter construct carrying the minimal promoter regions of the muscle dystrophin isoform. Consistent with this apparently repressive role, we found that, in muscle samples of dystrophinopathic female carriers, lncRNAs expression levels inversely correlate with those of muscle full length DMD isoforms. Overall these findings unveil an unprecedented complexity of the transcriptional pattern of the DMD locus and reveal that DMD lncRNAs may contribute to the orchestration and homeostasis of the muscle dystrophin expression pattern by either selective targeting and down-modulating the dystrophin promoter transcriptional activity.

c-MYC oncoprotein dictates transcriptional profiles of ATP-binding cassette transporter genes in Chronic Myelogenous Leukemia CD34+ hematopoietic progenitor cells

Resistance to chemotherapeutic agents remains one of the major impediments to a successful treatment of chronic myeloid leukemia (CML). Misregulation of the activity of a specific group of ATP-binding cassette transporters (ABC) is responsible for reducing the intracellular concentration of drugs in leukemic cells. Moreover, a consistent body of evidence also suggests that ABC transporters play a role in cancer progression beyond the efflux of cytotoxic drugs. Despite a large number of studies that investigated the function of the ABC transporters, little is known about the transcriptional regulation of the ABC genes. Here, we present data showing that the oncoprotein c-MYC is a direct transcriptional regulator of a large set of ABC transporters in CML. Furthermore, molecular analysis carried out in CD34+ hematopoietic cell precursors of 21 CML patients reveals that the overexpression of ABC transporters driven by c-MYC is a peculiar characteristic of the CD34+ population in CML and was not found either in the population of mononuclear cells from which they had been purified nor in CD34+ cells isolated from healthy donors. Finally, we describe how the methylation state of CpG islands may regulate the access of c-MYC to ABCG2 gene promoter, a well-studied gene associated with multidrug resistance in CML, hence, affecting its expression. Taken together, our findings support a model in which c-MYC–driven transcriptional events, combined with epigenetic mechanisms, direct and regulate the expression of ABC genes with possible implications in tumor malignancy and drug efflux in CML. Mol Cancer Res; 9(8); 1054–66. ©2011 AACR.

MYCN-mediated transcriptional repression in neuroblastoma: the other side of the coin

Neuroblastoma is the most common extra cranial solid tumor in childhood and the most frequently diagnosed neoplasm during the infancy. MYCN amplification and overexpression occur in about 25% of total neuroblastoma cases and this percentage increases at 30% in advanced stage neuroblastoma. So far, MYCN expression profile is still one of the most robust and significant prognostic markers for neuroblastoma outcome. MYCN is a transcription factor that belongs to the family of MYC oncoproteins, comprising c-MYC and MYCL genes. Deregulation of MYC oncoprotein expression is a crucial event involved in the occurrence of different types of malignant tumors. MYCN, as well as c-MYC, can heterodimerize with its partner MAX and activate the transcription of several target genes containing E-Box sites in their promoter regions. However, recent several lines of evidence have revealed that MYCN can repress at least as many genes as it activates, thus proposing a novel function of this protein in neuroblastoma biology. Whereas the mechanism by which MYCN can act as a transcriptional activator is relatively well known, very few studies has been done in the attempt to explain how MYCN can exert its transcription repression function. Here, we will review current knowledge about the mechanism of MYCN-mediated transcriptional repression and will emphasize its role as a repressor in the recruitment of a precise set of proteins to form complexes capable of down-regulating specific subsets of genes whose function is actively involved in apoptosis, cell differentiation, chemosensitivity, and cell motility. The finding that MYCN can also act as a repressor has widen our view on its role in oncogenesis and has posed the bases to search for novel therapeutic drugs that can specifically target its transcriptional repression function.