Myc oncoproteins are commonly upregulated in human cancers of different organ origins, stabilized by Aurora A, degraded through ubiquitin-proteasome pathway-mediated proteolysis, and exert oncogenic effects by modulating gene and protein expression. Histone deacetylases are emerging as targets for cancer therapy. Here we demonstrated that the class III histone deacetylase SIRT2 was upregulated by N-Myc in neuroblastoma cells and by c-Myc in pancreatic cancer cells, and that SIRT2 enhanced N-Myc and c-Myc protein stability and promoted cancer cell proliferation. Affymetrix gene array studies revealed that the gene most significantly repressed by SIRT2 was the ubiquitin-protein ligase NEDD4. Consistent with this finding, SIRT2 repressed NEDD4 gene expression by directly binding to the NEDD4 gene core promoter and deacetylating histone H4 lysine 16. Importantly, NEDD4 directly bound to Myc oncoproteins and targeted Myc oncoproteins for ubiquitination and degradation, and small-molecule SIRT2 inhibitors reactivated NEDD4 gene expression, reduced N-Myc and c-Myc protein expression, and suppressed neuroblastoma and pancreatic cancer cell proliferation. Additionally, SIRT2 upregulated and small-molecule SIRT2 inhibitors decreased Aurora A expression. Our data reveal a novel pathway critical for Myc oncoprotein stability, and provide important evidences for potential application of SIRT2 inhibitors for the prevention and therapy of Myc-induced malignancies. The Myc family of oncoproteins are commonly upregulated in human cancer. MYCN oncogene amplification and consequent N-Myc oncoprotein overexpression occur in 20-25% of neuroblastoma and correlate with a poor patient outcome. 1-3 MYC oncogene amplification occurs in 54% of human pancreatic cancer cell lines 4 and 33% of human primary pancreatic tumors, 5 and significant c-Myc oncoprotein overexpression is seen in B50% of human primary pancreatic tumors. 6 Stabilization and degradation of Myc oncoproteins are controlled by ordered phosphorylation at serine 62 (S62) and threonine 58 (T58) and consequent ubiquitinproteasome pathway-mediated proteolysis. 7-9 Aurora A interacts with both N-Myc and ubiquitin, and blocks ubiquitin-regulated N-Myc protein degradation. 8 Myc oncoproteins induce malignant transformation by binding to cognate DNA sequences and consequently modulating gene transcription 10-13 as well as by enhancing ribosome biogenesis and consequently upregulating protein expression, 14,15 leading to cell proliferation.Recruitment of histone deacetylase (HDACs) to gene promoters induces histone hypoacetylation and transcriptional repression, particularly of tumor suppressor genes. 16 In a comprehensive panel of normal cells, cancer cell lines, normal tissues, and primary tumors, global loss of monoacetylation of histone H4 at lysine 16 (H4K16) is seen only in cancer cells and is associated with early stages of tumorigenesis. 17 One of the HDACs that cause H4K16 deacetylation is the class III HDAC SIRT2, which shows a strong preference for acetylated H4K16. 18 Mo...

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.

Amplification of the MYCN oncogene predicts treatment resistance in childhood neuroblastoma. We used a MYC target gene signature that predicts poor neuroblastoma prognosis to identify the histone chaperone, FAcilitates Chromatin Transcription (FACT), as a crucial mediator of the MYC signal and a therapeutic target in the disease. FACT and MYCN expression created a forward feedback loop in neuroblastoma cells that was essential for maintaining mutual high expression. FACT inhibition by the small molecule curaxin compound, CBL0137, markedly reduced tumor initiation and progression in vivo. CBL0137 exhibited strong synergy with standard chemotherapy by blocking repair of DNA damage caused by genotoxic drugs, thus creating a synthetic lethal environment in MYCN-amplified neuroblastoma cells and suggesting a treatment strategy for MYCN-driven neuroblastoma.

Patients with neuroblastoma due to N-Myc oncogene amplification have a high frequency of tumor metastasis. However, it is not clear how N-Myc induces cell migration, invasion and metastasis. The histone demethylase JMJD1A activates gene transcription by demethylating the lysine 9 residue of histone H3 (H3K9) at target gene promoters. The long noncoding RNA MALAT1 induces lung cancer cell migration and plays a pivotal role in lung cancer metastasis. Here we demonstrated that N-Myc up-regulated the expression of JMJD1A in N-Myc oncogene-amplified human neuroblastoma cells by directly binding to the JMJD1A gene promoter. Affymetrix microarray studies revealed that the gene second most significantly up-regulated by JMJD1A was MALAT1. Consistent with this finding, RT-PCR and chromatin immunoprecipitation assays showed that JMJD1A bound to the MALAT1 gene promoter and demethylated histone H3K9 at the MALAT1 gene promoter. Moreover, JMJD1A and MALAT1 induced, while the small molecule JMJD1A inhibitor DMOG suppressed, neuroblastoma cell migration and invasion. Taken together, our data identify a novel pathway through which N-Myc causes neuroblastoma cell migration and invasion, and provide important evidence for further development of more potent JMJD1A/MALAT1 inhibitors for the prevention of tumor metastasis.