In human neuroblastoma, amplification of the MYCN gene predicts poor prognosis and resistance to therapy. In a shRNA screen of genes that are highly expressed in MYCN-amplified tumors, we have identified AURKA as a gene that is required for the growth of MYCN-amplified neuroblastoma cells but largely dispensable for cells lacking amplified MYCN. Aurora A has a critical function in regulating turnover of the N-Myc protein. Degradation of N-Myc requires sequential phosphorylation by cyclin B/Cdk1 and Gsk3. N-Myc is therefore degraded during mitosis in response to low levels of PI3-kinase activity. Aurora A interacts with both N-Myc and the SCF(Fbxw7) ubiquitin ligase that ubiquitinates N-Myc and counteracts degradation of N-Myc, thereby uncoupling N-Myc stability from growth factor-dependent signals.

The MYC proto-oncogene encodes a transcription factor that has been implicated in the genesis of many human tumours. Here, we used a bar-code short hairpin RNA (shRNA) screen to identify multiple genes that are required for MYC function. One of these genes encodes USP28, an ubiquitin-specific protease. USP28 is required for MYC stability in human tumour cells. USP28 binds to MYC through an interaction with FBW7alpha, an F-box protein that is part of an SCF-type ubiquitin ligase. Therefore, it stabilizes MYC in the nucleus, but not in the nucleolus, where MYC is degraded by FBW7gamma. High expression levels of USP28 are found in colon and breast carcinomas, and stabilization of MYC by USP28 is essential for tumour-cell proliferation.

The Myc oncoprotein forms a binary activating complex with its partner protein, Max, and a ternary repressive complex that, in addition to Max, contains the zinc finger protein Miz1. Here we show that the E3 ubiquitin ligase HectH9 ubiquitinates Myc in vivo and in vitro, forming a lysine 63-linked polyubiquitin chain. Miz1 inhibits this ubiquitination. HectH9-mediated ubiquitination of Myc is required for transactivation of multiple target genes, recruitment of the coactivator p300, and induction of cell proliferation by Myc. HectH9 is overexpressed in multiple human tumors and is essential for proliferation of a subset of tumor cells. Our results suggest that site-specific ubiquitination regulates the switch between an activating and a repressive state of the Myc protein, and they suggest a strategy to interfere with Myc function in vivo.

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R e s e a R c h a R t i c l e3 4 0 7 jci.org Volume 124 Number 8 August 2014 IntroductionThe intestine is made up of repetitive units that consist of a differentiated compartment (the villus) and a proliferative compartment (the crypt). Intestinal stem cells are located in the crypt (1, 2) and produce rapidly proliferating daughter cells, the transit amplifying cells, which subsequently differentiate into 2 main epithelial lineages. The absorptive lineage is composed of all enterocytes, while the secretory lineage is composed of goblet cells (secreting protective mucins), enteroendocrine cells (secreting hormones like serotonin or secretin), and Paneth cells (3, 4). Whether transit amplifying cells differentiate along an absorptive or a secretory lineage is regulated by the Notch pathway (5). Engagement of Notch receptors by Notch ligands, such as Delta or Jagged, induces proteolytic cleavage of the receptor by γ-secretase. The cleaved NOTCH1 receptor (NICD1) translocates into the nucleus, resulting in the formation of an active transcriptional complex composed of RBPJκ (also known as CSL or CBF1) and NICD1. Notch signal activation induces hairy/enhancer of split (Hes) gene expression. Ablation of Notch signaling via genetic deletion of Rbpj results in secretory cell expansion (6). Conversely, in transgenic mice overexpressing NICD1, goblet cells are absent, and the proliferative compartment is expanded (7).The Wnt signaling pathway is a key regulator of intestinal stem cell homeostasis (8), and 2 of the target genes induced by Wnt signaling, c-MYC and c-JUN, encode transcription factors that have oncogenic potential (9, 10). Consequently, aberrant activation of the adenomatous polyposis coli/β-catenin/T cell factor (APC/ β-catenin/TCF) pathway is an initiating event in the majority of human colorectal cancers (11).c-MYC is a transcription factor with key functions in cell differentiation and cancer development (12)(13)(14). In the intestine, c-MYC is required for the altered proliferation and differentiation induced by APC inactivation (15-18). c-MYC is a highly labile protein, and at least 2 ubiquitin ligases, SKP2 and FBW7, can target it for proteasomal degradation (19-21). c-MYC ubiquitination is antagonized by the deubiquitinase USP28, which "piggybacks" on FBW7 and stabilizes c-MYC protein (22). Thus, an E3 ubiquitin ligase and a deubiquitinase, FBW7 and USP28, are together recruited to substrates (22), and a cycle of deubiquitination and ubiquitination controls c-MYC stability.Genomic data from human cancers suggest that most colorectal cancer mutations converge on c-MYC misregulation (23). Due to its key role in tumorigenesis, much recent research has been directed to finding ways to target c-MYC function (24-29). Dominant-negative approaches targeting c-MYC function impair intestinal tumor formation, and c-Myc heterozygous mice show reduced tumor development in the Apc min/+ model (16,17). Transgenic expression of a dominant-negative allele of Myc, OmoMyc, has provided proof of principle that targeting ...

The SCFFbw7 ubiquitin ligase mediates growth-factor-regulated turnover of the Myc oncoprotein. Here we show that SCFβ-TrCP binds to Myc by means of a characteristic phosphodegron and ubiquitylates Myc; this results in enhanced Myc stability. SCFFbw7 and SCFβ-TrCP can exert these differential effects through polyubiquitylation of the amino terminus of Myc. Whereas SCFFbw7 with the Cdc34 ubiquitin-conjugating enzyme specifically requires lysine 48 (K48) of ubiquitin, SCFβ-TrCP uses the UbcH5 ubiquitin-conjugating enzyme to form heterotypic polyubiquitin chains on Myc. Ubiquitylation of Myc by SCFβ-TrCP is required for Myc-dependent acceleration of cell cycle progression after release from an arrest in S phase. Therefore, alternative ubiquitylation events at the N terminus can lead to the ubiquitylation-dependent stabilization of Myc.

The cellular levels of the Myc oncoprotein are critical determinants of cell proliferation, cell growth and apoptosis and are tightly regulated by external growth factors. Levels of Myc oncoprotein also decline in response to intracellular stress signals such as DNA damage. We show here that this decline is in part due to proteasomal degradation and that it is mediated by the Fbw7 ubiquitin ligase. We have shown previously that the ubiquitin-specific protease Usp28, binds to the nucleoplasmic isoform of Fbw7, Fbw7α, and counteracts its function in mammalian cells. Usp28 dissociates from Fbw7α in response to UV irradiation, providing a mechanism how Fbw7-mediated degradation of Myc is enhanced upon DNA damage. Our data extend previous observations that link Myc function to the cellular response to DNA damage.

Deregulated expression of MYC is a driver of colorectal carcinogenesis, necessitating novel strategies to inhibit MYC function. The ubiquitin ligase HUWE1 (HECTH9, ARF-BP1, MULE) associates with both MYC and the MYC-associated protein MIZ1. We show here that HUWE1 is required for growth of colorectal cancer cells in culture and in orthotopic xenograft models. Using high-throughput screening, we identify small molecule inhibitors of HUWE1, which inhibit MYC-dependent transactivation in colorectal cancer cells, but not in stem and normal colon epithelial cells. Inhibition of HUWE1 stabilizes MIZ1. MIZ1 globally accumulates on MYC target genes and contributes to repression of MYC-activated target genes upon HUWE1 inhibition. Our data show that transcriptional activation by MYC in colon cancer cells requires the continuous degradation of MIZ1 and identify a novel principle that allows for inhibition of MYC function in tumor cells.See also: FX Schaub & JL Cleveland (December 2014)