The c-Myc oncoprotein is essential for normal cell growth and proliferation. However, overexpression of c-Myc occurs in most human cancers. Thus, its level and activity must be tightly regulated during normal cell homeostasis. The ubiquitination-proteasome system plays a key role in controlling c-Myc levels and activity. c-Myc normally undergoes rapid ubiquitin-dependent proteolysis, but it is transiently stabilized by key phosphorylation events in response to growth signals. Phosphorylation of Serine 62 (S62) stabilizes c-Myc, whereas phosphorylation of Threonine 58 (T58) promotes c-Myc ubiquitination by the SCFFbw7 ubiquitin ligase and proteasomal degradation, mainly in the nucleolus. Like other post-translational modifications, ubiquitination can be reversed by the action of deubiquitinating enzymes (DUBs). While several ubiquitin ligases have been identified for c-Myc, only one DUB, USP28, has been reported to target c-Myc. We have recently discovered that the nucleolar deubiquitinating enzyme USP36 is a novel c-Myc regulator. USP36 binds to c-Myc and deubiquitinates c-Myc in cells and in vitro. Overexpression of wild-type USP36, but not its catalytic-inactive C131A mutant, stabilizes c-Myc and enhances c-Myc-driven transcription. Knockdown of USP36 reduces c-Myc levels and drastically suppresses cell proliferation. Importantly, USP36 interacts with the nucleolar Fbw7? and abolishes Fbw7?-mediated c-Myc degradation. In contrast, USP28 antagonizes Fbw7?-mediated c-Myc degradation. Since the bulk of c-Myc is degraded in the nucleolus, our discovery leads to the novel hypothesis that USP36 functions as a crucial regulator of c-Myc by deubiquitinating c-Myc in the nucleolus. Interestingly, we found that USP36 itself is a c-Myc target gene, suggesting that USP36 and c-Myc form a positive feed-forward regulatory loop. To gain further insight into the role of USP36 in the regulation of c-Myc protein stability, activity and oncogenicity, we will investigate the molecular and biochemical mechanisms underlying the regulation of c-Myc by USP36 in Aim 1, including how USP36 interplays with Fbw7? to regulate c-Myc in the nucleolus, whether it interplays with USP28 in the dynamic control of c-Myc ubiquitination, and the importance of c-Myc-USP36 feed-forward regulation. We will elucidate the functional consequences of USP36 regulation of c-Myc in cells in Aim 2 by analyzing whether USP36 regulates c-Myc binding and turnover at target gene promoters, whether it promotes c-Myc-dependent ribosome biogenesis, and whether it promotes c-Myc's oncogenic potential in cells and in vivo. Finally, we will elucidate whether USP36 is a therapeutic target using cell based and mouse models as proposed in Aim 3, including the investigation of USP36 deregulation in human breast cancers, whether deletion of USP36 inhibits c-Myc-driven mammary tumorigenesis in mice, and high-throughput screening of small molecule inhibitors for USP36. Achieving these goals will provide critical insight into how c-Myc is properly regulated by dynamic ubiquitination and deubiquitination, how deregulation of this dynamic contributes to tumorigenesis, and how USP36 can be targeted in human cancers.

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Deregulated high-level expression of the c-Myc protein contributes significantly to cancer formation and progression. This study aims at elucidating the molecular mechanisms controlling c-Myc protein levels and activity by the deubiquitinating enzyme USP36, which could yield important insight into how to target USP36 for cancer therapy.

National Institute of Health (NIH)
National Cancer Institute (NCI)
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Molecular Oncogenesis Study Section (MONC)
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Strasburger, Jennifer
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Oregon Health and Science University
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DeVine, Tiffany; Sears, Rosalie C; Dai, Mu-Shui (2018) The ubiquitin-specific protease USP36 is a conserved histone H2B deubiquitinase. Biochem Biophys Res Commun 495:2363-2368
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