Jessica Koach

The histone deacetylase SIRT2 stabilizes Myc oncoproteins.

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.

Vaccination of Cattle with a CpG Oligodeoxynucleotide-Formulated Mycobacterial Protein Vaccine and Mycobacterium bovis BCG Induces Levels of Protection against Bovine Tuberculosis Superior to Those Induced by Vaccination with BCG Alone

ABSTRACT The development of a subunit protein vaccine for bovine tuberculosis which could be used either in combination with Mycobacterium bovis BCG (to improve the efficacy of that vaccine) or alone would offer significant advantages over currently available strategies. A study was conducted with cattle to determine the protective efficacy of a strategy based on concurrent immunization with an M. bovis culture filtrate (CFP) vaccine and BCG compared to vaccination with either vaccine alone. One group of calves (10 animals per group) was vaccinated subcutaneously with CFP formulated with Emulsigen and combined with a CpG oligodeoxynucleotide (ODN). A second group was vaccinated with both the CFP vaccine and BCG injected at adjacent sites (CFP-BCG). One further group was vaccinated subcutaneously with BCG, while another group served as nonvaccinated control animals. Vaccination with CFP-BCG induced levels of antigen-specific gamma interferon (IFN-γ) and interleukin-2 (IL-2) in whole-blood cultures that were higher than those induced by vaccination with BCG alone. The combination of CFP and BCG did not enhance the production of antibodies to M. bovis CFP compared to vaccination with CFP alone. Vaccination with CFP alone led to delayed antigen-specific IFN-γ and IL-2 responses. Vaccination with CFP-BCG induced a high level of protection against an intratracheal challenge with virulent M. bovis, based on a significant enhancement of six pathological and microbiological parameters of protection compared with the nonvaccinated group. In contrast, vaccination with BCG alone induced a significant enhancement of protection in only one parameter, while CFP alone induced no protection. These results suggest that a combination of a CpG ODN-formulated protein vaccine and BCG offers better protection against bovine tuberculosis than does BCG alone.

TRIM16 acts as an E3 ubiquitin ligase and can heterodimerize with other TRIM family members.

The TRIM family of proteins is distinguished by its tripartite motif (TRIM). Typically, TRIM proteins contain a RING finger domain, one or two B-box domains, a coiled-coil domain and the more variable C-terminal domains. TRIM16 does not have a RING domain but does harbour two B-box domains. Here we showed that TRIM16 homodimerized through its coiled-coil domain and heterodimerized with other TRIM family members; TRIM24, Promyelocytic leukaemia (PML) protein and Midline-1 (MID1). Although, TRIM16 has no classic RING domain, three-dimensional modelling of TRIM16 suggested that its B-box domains adopts RING-like folds leading to the hypothesis that TRIM16 acts as an ubiquitin ligase. Consistent with this hypothesis, we demonstrated that TRIM16, devoid of a classical RING domain had auto-polyubiquitination activity and acted as an E3 ubiquitin ligase in vivo and in vitro assays. Thus via its unique structure, TRIM16 possesses both heterodimerization function with other TRIM proteins and also has E3 ubiquitin ligase activity.

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.

Therapeutic targeting of the MYC signal by inhibition of histone chaperone FACT in neuroblastoma

Histone chaperone FACT acts in a positive feedback loop with MYCN and is a therapeutic target in neuroblastoma. Uncovering the FACTs in neuroblastoma Neuroblastoma is a common pediatric cancer of the nervous system. It is often difficult to treat, and tumors with amplifications of the MYC oncogene are particularly aggressive. Carter et al. have identified a histone chaperone called FACT as a mediator of MYC signaling in neuroblastoma and demonstrated its role in a feedback loop that allows tumor cells to maintain a high expression of both MYC and FACT. The authors then used curaxins, which are drugs that inhibit FACT, to break the vicious cycle. They demonstrated that curaxins work in synergy with standard genotoxic chemotherapy to kill cancer cells and treat neuroblastoma in mouse models. 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 FACT (facilitates chromatin transcription) 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.

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

The estrogen-responsive B box protein (EBBP) restores retinoid sensitivity in retinoid-resistant cancer cells via effects on histone acetylation

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) beta(2) 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 RARbeta2 transcriptional regulator in the retinoid signal. Here we examined the mechanism of the EBBP effect on the retinoid anticancer signal. We assessed retinoid-responsive RARbeta2 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 RARbeta2 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 (betaRARE) 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 RARbeta2 and CYP26A1, by modifying histone acetylation in retinoid-resistant cancer cells, and, is an important target for drug discovery in retinoid-resistant cancers.

Synthesis of retinoid enhancers based on 2-aminobenzothiazoles for anti-cancer therapy.

Indole-3-amides and dipeptides were produced from 2-aminobenzothiazoles using the PyBop peptide coupling reagent. These analogues were tested in anti-cancer cell viability assays against SH-SY5Y neuroblastoma and MDA-MB-231 breast adenocarcinoma cell lines, and were found to exhibit cytotoxic activities at concentrations ranging from 0.1 to 20μM. These compounds were also found to act additively with a low dosage of 13-cis-retinoic acid in neuroblastoma cells. Then, using neuroblastoma cells transfected to stably overexpress the RARβ(2) gene, a SAR was developed for the indole-3-amides. Real-time PCR was also used to demonstrate their RARβ(2) agonistic activity.

MYCN promotes neuroblastoma malignancy by establishing a regulatory circuit with transcription factor AP4

Amplification of the MYCN oncogene, a member of the MYC family of transcriptional regulators, is one of the most powerful prognostic markers identified for poor outcome in neuroblastoma, the most common extracranial solid cancer in childhood. While MYCN has been established as a key driver of malignancy in neuroblastoma, the underlying molecular mechanisms are poorly understood. Transcription factor activating enhancer binding protein-4 (TFAP4) has been reported to be a direct transcriptional target of MYC. We show for the first time that high expression of TFAP4 in primary neuroblastoma patients is associated with poor clinical outcome. siRNA-mediated suppression of TFAP4 in MYCN-expressing neuroblastoma cells led to inhibition of cell proliferation and migration. Chromatin immunoprecipitation assay demonstrated that TFAP4 expression is positively regulated by MYCN. Microarray analysis identified genes regulated by both MYCN and TFAP4 in neuroblastoma cells, including Phosphoribosyl-pyrophosphate synthetase-2 (PRPS2) and Syndecan-1 (SDC1), which are involved in cancer cell proliferation and metastasis. Overall this study suggests a regulatory circuit in which MYCN by elevating TFAP4 expression, cooperates with it to control a specific set of genes involved in tumor progression. These findings highlight the existence of a MYCN-TFAP4 axis in MYCN-driven neuroblastoma as well as identifying potential therapeutic targets for aggressive forms of this disease.