Targeting the Oncogenic E3 Ligase Skp2 in Prostate and Breast Cancer Cells with a Novel Energy Restriction-Mimetic Agent

PLOS ONE, Dec 2019

Substantial evidence supports the oncogenic role of the E3 ubiquitin ligase S-phase kinase-associated protein 2 (Skp2) in many types of cancers through its ability to target a broad range of signaling effectors for ubiquitination. Thus, this oncogenic E3 ligase represents an important target for cancer drug discovery. In this study, we report a novel mechanism by which CG-12, a novel energy restriction-mimetic agent (ERMA), down-regulates the expression of Skp2 in prostate cancer cells. Pursuant to our previous finding that upregulation of β-transducin repeat-containing protein (β-TrCP) expression represents a cellular response in cancer cells to ERMAs, including CG-12 and 2-deoxyglucose, we demonstrated that this β-TrCP accumulation resulted from decreased Skp2 expression. Evidence indicates that Skp2 targets β-TrCP for degradation via the cyclin-dependent kinase 2-facilitated recognition of the proline-directed phosphorylation motif 412SP. This Skp2 downregulation was attributable to Sirt1-dependent suppression of COP9 signalosome (Csn)5 expression in response to CG-12, leading to increased cullin 1 neddylation in the Skp1-cullin1-F-box protein complex and consequent Skp2 destabilization. Moreover, we determined that Skp2 and β-TrCP are mutually regulated, providing a feedback mechanism that amplifies the suppressive effect of ERMAs on Skp2. Specifically, cellular accumulation of β-TrCP reduced the expression of Sp1, a β-TrCP substrate, which, in turn, reduced Skp2 gene expression. This Skp2-β-TrCP-Sp1 feedback loop represents a novel crosstalk mechanism between these two important F-box proteins in cancer cells with aberrant Skp2 expression under energy restriction, which provides a proof-of-concept that the oncogenic Csn5/Skp2 signaling axis represents a “druggable” target for this novel ERMA.

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Targeting the Oncogenic E3 Ligase Skp2 in Prostate and Breast Cancer Cells with a Novel Energy Restriction-Mimetic Agent

et al. (2012) Targeting the Oncogenic E3 Ligase Skp2 in Prostate and Breast Cancer Cells with a Novel Energy Restriction-Mimetic Agent. PLoS ONE 7(10): e47298. doi:10.1371/journal.pone.0047298 Targeting the Oncogenic E3 Ligase Skp2 in Prostate and Breast Cancer Cells with a Novel Energy Restriction- Mimetic Agent Shih Chen 0 Shuo Wei 0 Po-Chen Chu 0 Hsiao-Ching Chuang 0 Wen-Chun Hung 0 Samuel K. Kulp 0 Ching- 0 Natasha Kyprianou, University of Kentucky College of Medicine, United States of America 0 1 Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University , Columbus , Ohio, United States of America, 2 National Institute of Cancer Research, National Health Research Institute , Zhunan, Miaoli County , Taiwan , 3 Institute of Basic Medical Sciences, National Cheng-Kung University , Tainan , Taiwan Substantial evidence supports the oncogenic role of the E3 ubiquitin ligase S-phase kinase-associated protein 2 (Skp2) in many types of cancers through its ability to target a broad range of signaling effectors for ubiquitination. Thus, this oncogenic E3 ligase represents an important target for cancer drug discovery. In this study, we report a novel mechanism by which CG-12, a novel energy restriction-mimetic agent (ERMA), down-regulates the expression of Skp2 in prostate cancer cells. Pursuant to our previous finding that upregulation of b-transducin repeat-containing protein (b-TrCP) expression represents a cellular response in cancer cells to ERMAs, including CG-12 and 2-deoxyglucose, we demonstrated that this bTrCP accumulation resulted from decreased Skp2 expression. Evidence indicates that Skp2 targets b-TrCP for degradation via the cyclin-dependent kinase 2-facilitated recognition of the proline-directed phosphorylation motif 412SP. This Skp2 downregulation was attributable to Sirt1-dependent suppression of COP9 signalosome (Csn)5 expression in response to CG12, leading to increased cullin 1 neddylation in the Skp1-cullin1-F-box protein complex and consequent Skp2 destabilization. Moreover, we determined that Skp2 and b-TrCP are mutually regulated, providing a feedback mechanism that amplifies the suppressive effect of ERMAs on Skp2. Specifically, cellular accumulation of b-TrCP reduced the expression of Sp1, a b-TrCP substrate, which, in turn, reduced Skp2 gene expression. This Skp2-b-TrCP-Sp1 feedback loop represents a novel crosstalk mechanism between these two important F-box proteins in cancer cells with aberrant Skp2 expression under energy restriction, which provides a proof-of-concept that the oncogenic Csn5/Skp2 signaling axis represents a ''druggable'' target for this novel ERMA. - Funding: This work was supported by Public Health Service grant CA112250 from the National Cancer Institute to C.S.C. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. The ubiquitin-proteasome system plays a pivotal role in regulating key cellular functions through targeted degradation of regulatory proteins, which is initiated by ubiquitination, followed by proteolysis via the 26S proteasome complex. The targeted ubiquitination is mediated through the concerted action of three enzymes: E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme, and E3 ubiquitin ligase. Among various E3 ubiquitin ligases, the Skp1-Cul1-F-box protein (SCF) complex represents the largest family, which has received much attention because of its intimate role in regulating cell cycle progression by facilitating targeted degradation of cell cycle-regulatory proteins [1]. The specificity of the SCF ubiquitin ligase for its target proteins is conferred by interchangeable substrate-recognizing subunits, i.e., F-box proteins. More than 70 F-box proteins, each specific for a distinct set of substrates, have been identified in human cells, which provides the basis for the targeted degradation of diverse regulatory proteins during cell cycle progression [2]. Some of these F-box proteins have received much attention in light of their involvement in tumorigenesis, among which S-phase kinaseassociated protein (Skp)2 and b-transducin repeat-containing protein (b-TrCP) are especially noteworthy [2]. Substantial evidence indicates that Skp2 acts as an oncoprotein by targeting a wide range of signaling effectors, such as the tumor suppressor p27 [3], for degradation. More recently, it was demonstrated that Skp2 facilitates the activation of Akt through ubiquitination downstream of ErbB receptor signaling in Her2-positive breast cancer [4], and that Skp2, by triggering NSB1 ubiquitination, represents a key component for the Mre11/Rad50/NBS1 complex-mediated activation of ATM in response to DNA double-strand breaks [5]. Consequently, its overexpression has been correlated with poor prognosis in many types of cancers [2]. In contrast, b-TrCP plays an (...truncated)


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Shuo Wei, Po-Chen Chu, Hsiao-Ching Chuang, Wen-Chun Hung, Samuel K. Kulp, Ching-Shih Chen. Targeting the Oncogenic E3 Ligase Skp2 in Prostate and Breast Cancer Cells with a Novel Energy Restriction-Mimetic Agent, PLOS ONE, 2012, 10, DOI: 10.1371/journal.pone.0047298