Withaferin A activates TRIM16 for its anti‐cancer activity in melanoma

Although selective BRAF inhibitors and novel immunotherapies have improved short-term treatment responses in metastatic melanoma patients, acquired resistance to these therapeutics still represent a major challenge in clinical practice. In this study, we evaluated the efficacy of Withaferin A (WFA), derived from the medicinal plant Withania Somnifera, as a novel therapeutic agent for the treatment of melanoma. WFA showed selective toxicity to melanoma cells compared to non-malignant cells. WFA induced apoptosis, significantly reduced cell proliferation and inhibited migration of melanoma cells. We identified that repression of the tumour suppressor TRIM16 diminished WFA cytotoxicity, suggesting that TRIM16 was in part responsible for the cytotoxic effects of WFA in melanoma cells. Together our data indicates that WFA has potent cytopathic effects on melanoma cells through TRIM16, suggesting a potential therapeutic application of WFA in the disease

MYCN-targeting miRNAs are predominantly downregulated during MYCN-driven neuroblastoma tumor formation

MYCN is a transcription factor that plays key roles in both normal development and cancer. In neuroblastoma, MYCN acts as a major oncogenic driver through pleiotropic effects controlled by multiple protein encoding genes as well as microRNAs (miRNAs). MYCN activity is tightly regulated at the level of transcription and protein stability through various mechanisms. Like most genes, MYCN is further controlled by miRNAs, but the full complement of all miRNAs implicated in this process has not been determined through an unbiased approach. To elucidate the role of miRNAs in regulation of MYCN, we thus explored the MYCN-miRNA interactome to establish miRNAs controlling MYCN expression levels. We combined results from an unbiased and genome-wide high-throughput miRNA target reporter screen with miRNA and mRNA expression data from patients and a murine neuroblastoma progression model. We identified 29 miRNAs targeting MYCN, of which 12 miRNAs are inversely correlated with MYCN expression or activity in neuroblastoma tumor tissue. The majority of MYCN-targeting miRNAs in neuroblastoma showed a decrease in expression during murine MYCN-driven neuroblastoma tumor development. Therefore, we provide evidence that MYCN-targeting miRNAs are preferentially downregulated in MYCN-driven neuroblastoma, suggesting that MYCN negatively controls the expression of these miRNAs, to safeguard its expression

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

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

The long noncoding RNA lncNB1 promotes tumorigenesis by interacting with ribosomal protein RPL35

The majority of patients with neuroblastoma due to MYCN oncogene amplification and consequent N-Myc oncoprotein over-expression die of the disease. Here our analyses of RNA sequencing data identify the long noncoding RNA lncNB1 as one of the transcripts most over-expressed in MYCN-amplified, compared with MYCN-non-amplified, human neuroblastoma cells and also the most over-expressed in neuroblastoma compared with all other cancers. lncNB1 binds to the ribosomal protein RPL35 to enhance E2F1 protein synthesis, leading to DEPDC1B gene transcription. The GTPase-activating protein DEPDC1B induces ERK protein phosphorylation and N-Myc protein stabilization. Importantly, lncNB1 knockdown abolishes neuroblastoma cell clonogenic capacity in vitro and leads to neuroblastoma tumor regression in mice, while high levels of lncNB1 and RPL35 in human neuroblastoma tissues predict poor patient prognosis. This study therefore identifies lncNB1 and its binding protein RPL35 as key factors for promoting E2F1 protein synthesis, N-Myc protein stability and N-Myc-driven oncogenesis, and as therapeutic targets

TRIM16 homodimerizes through its coiled-coil domain

<p>. (A) TRIM16-GFP domain deletion plasmids. (B) Co-transfection of TRIM16-GFP and TRIM16-myc-His in HEK293 cells and subsequent immunoprecipitation by anti-myc antibody (Ab) and Western blot with anti-GFP antibody. Whole cell extract (WCE) used as total input. (C) TRIM16 homodimerizes through its coiled-coil domain. GFP deletion mutants were co-transfected with the TRIM16-myc-His vector. Anti-GFP antibody was used to pull down proteins binding the GFP tagged proteins and the TRIM16-myc-His was used to detect self-association via its different tag (right panel). Transfection efficiency was confirmed (left panel). TRIM16-GFP mutants were efficiently pulled down (middle panel). * non-specific bands, # refer to text.</p

TRIM16 can heterodimerize with MID1, TRIM24 and PML.

<p>(A) Schematic structures of TRIM proteins used in heterodimerization studies. (B) TRIM16 binds MID1. Co-transfection of MID1-GFP and TRIM16-myc-His in HEK293 cells and subsequent immunoprecipitation by anti-myc antibody and Western blot with anti-GFP antibody. (C) MID1 was pulled down via its GFP antibody and a Western blot was performed to detect TRIM16-myc-His in the immunoprecipitated protein complex. (D) Whole cell lysates (WCL) of HEK293 cells transfected with empty vector (EV) or TRIM16-GFP were immunopreciptated with anti-GFP antibody. An anti-PML antibody was used to detect PML as a binding partner of TRIM16. (E) Lysates containing both TRIM16-GFP and TRIM24-His proteins were immunopreciptated with anti-GFP antibody. Anti-His antibody was used to detect the presence of TRIM24 in the TRIM16-associated complex.</p

Amino acid sequence comparison of TRIM16 and MID1 and modeling of TRIM16 B-boxes.

<p>The conserved residues in the zinc binding regions are in bold underlined type and are; cysteine; C, histidine; H, alanine; A aspartic acid; D. (<b>A</b>) TRIM16 and MID1 share the zinc binding consensus sequence for B-box1. (B) TRIM16 and MID1 share the Zinc binding consensus sequence for B-box2. (C/D) Modeling of B-boxes reveals zinc binding capability. Superimposition of the alpha-carbon backbone of the B-boxes from the MDM1 NMR structure (purple) and the homology model of TRIM16 (blue-grey). These two structures overlay with an average root-mean-square deviation of 0.4 Å.</p