Targeted cancer therapy relies on a thorough validation of cancer targets. Our long-range goal is to discover a novel class of anticancer drugs that selectively target one type of E3 ubiquitin ligase, which is activated in human cancer. Selective targeting one ubiquitin ligase pathway will reduce normal cell toxicity associated with overall inhibition of protein degradation, as seen in Velcade (also known as Bortezomib or PS-341), the first (and only) class of general proteasome inhibitor, approved by FDA for the treatment of relapsed/refractory multiple myeloma and mantle cell lymphoma. To this end, we have focused on SCF (Skp1-Cullin-F-box proteins) E3 ubiquitin ligases, also known as CRLs (Cullin-RING ubiquitin ligases). SCF E3 ligases, the largest E3 ligase family consisting of Skp1, cullins, F-box proteins, and a RING protein, ROC or RBX, promotes the ubiquitination of a subset of key regulatory proteins for targeted degradation, thus governing important biological processes, including cell cycle progression, signal transduction and DNA replication. While cullin-ROC constitutes the core ligase component, cullin needs to be neddylated via Nedd8-Activating Enzyme (NAE) for its enzymatic activity. Our strong preliminary data showed that ROC1 is over-expressed in a number of human cancers, and inactivation of SCF E3 ubiquitin ligase activity via ROC1 siRNA silencing or by an NAE inhibitor, MLN4924, triggers DNA double strand breaks (DSB) and the DNA damage response (DDR), and induces tumor cell killing via autophagy, senescence and apoptosis. Inhibition of SCF E3 ligase also enhances radiation-induced DDR, leading to radiosensitization. The objective of this proposed study is to elucidate the underlying mechanisms by which inactivation of ROC1-SCF E3 ligase triggers these biochemical and biological changes, leading to various types of cell death, and to validate ROC1-SCF E3 ligase as an anticancer and radiosensitizing target. Our central hypothesis is that inactivation of ROC1-SCF E3 ubiquitin ligase causes accumulation of several key substrates, which triggers DSB and DDR and induces cell death via autophagy, senescence and apoptosis in a sequential or parallel order. These triggered cell killing mechanisms could also enhance radiation effects, leading to radiosensitization of cancer cells.
Three specific aims are proposed 1) to determine how inactivation of ROC1-SCF E3 ligase triggers DNA damage and DDR, and induces cell death via different mechanisms;2) to determine how inactivation of ROC1-SCF E3 ligase blocks mTOR to induce autophagy;and 3) to validate ROC1-SCF E3 ligase as a novel radiosensitizing target. IMPACT: This work is highly innovative and of significant impact with translational value by validating SCF E3 ligase as an anticancer target and by paving the ground for future development of MLN4924 or its analogues as a novel class of radiosensitizers.

Public Health Relevance

Proteasome inhibitor, Velcade is used in the clinic for the treatment of relapsed/refractory multiple myeloma. The drug is in general toxic to normal cells due to its overall inhibition of protein degradation. One way to reduce toxic side-effect is to inhibit a particular E3 ligase that is activated in cancer cells. This study will validate SCF E3 ubiquitin ligase as an anticancer and radiosensitizing target and MLN4924, a newly discovered SCF E3 ligase inhibitor, as a tumor-selective and radiosensitizing agent for cancer therapy.

National Institute of Health (NIH)
National Cancer Institute (NCI)
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Basic Mechanisms of Cancer Therapeutics Study Section (BMCT)
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Bernhard, Eric J
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University of Michigan Ann Arbor
Schools of Medicine
Ann Arbor
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Zhao, Yongchao; Tan, Mingjia; Liu, Xia et al. (2018) Inactivation of ribosomal protein S27-like confers radiosensitivity via the Mdm2-p53 and Mdm2-MRN-ATM axes. Cell Death Dis 9:145
Zhou, Lisha; Zhang, Wenjuan; Sun, Yi et al. (2018) Protein neddylation and its alterations in human cancers for targeted therapy. Cell Signal 44:92-102
Zhou, Weihua; Xu, Jie; Tan, Mingjia et al. (2018) UBE2M Is a Stress-Inducible Dual E2 for Neddylation and Ubiquitylation that Promotes Targeted Degradation of UBE2F. Mol Cell 70:1008-1024.e6
Zhou, Weihua; Xu, Jie; Li, Haomin et al. (2017) Neddylation E2 UBE2F Promotes the Survival of Lung Cancer Cells by Activating CRL5 to Degrade NOXA via the K11 Linkage. Clin Cancer Res 23:1104-1116
Li, Hua; Zhou, Weihua; Li, Lihui et al. (2017) Inhibition of Neddylation Modification Sensitizes Pancreatic Cancer Cells to Gemcitabine. Neoplasia 19:509-518
Xu, Jie; Zhou, Weihua; Yang, Fei et al. (2017) The ?-TrCP-FBXW2-SKP2 axis regulates lung cancer cell growth with FBXW2 acting as a tumour suppressor. Nat Commun 8:14002
Kuang, Peng; Tan, Mingjia; Zhou, Weihua et al. (2016) SAG/RBX2 E3 ligase complexes with UBCH10 and UBE2S E2s to ubiquitylate ?-TrCP1 via K11-linkage for degradation. Sci Rep 6:37441
Zhang, Qiang; Karnak, David; Tan, Mingjia et al. (2016) FBXW7 Facilitates Nonhomologous End-Joining via K63-Linked Polyubiquitylation of XRCC4. Mol Cell 61:419-433
Lan, Huiyin; Tang, Zaiming; Jin, Hongchuan et al. (2016) Neddylation inhibitor MLN4924 suppresses growth and migration of human gastric cancer cells. Sci Rep 6:24218
Zhou, Xiaochen; Tan, Mingjia; Nyati, Mukesh K et al. (2016) Blockage of neddylation modification stimulates tumor sphere formation in vitro and stem cell differentiation and wound healing in vivo. Proc Natl Acad Sci U S A 113:E2935-44

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