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    2020-08-12


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    the role of SPOP in mediating the stemness and pluripotency of PCa stem CCK-8 and ESCs remains largely unknown.
    In this study, we report that SPOP inhibits the self-renewal and stem-like characteristics of PCa via the ubiquitin-depen-dent degradation of NANOG. The cancer-associated NANOG S68Y mutant is refractory for SPOP-mediated degradation since NANOG Ser68 is required for the direct interaction between SPOP and NANOG. In parallel, AMPK activation promotes the NANOG degradation by blocking the binding of NANOG to BRAF that phosphorylates NANOG at Ser68. Thus, our study uncovers a de novo regulation mechanism of NANOG stability dictated by SPOP-induced degradation, which is abrogated by the phosphorylation of NANOG at Ser68 and thereby acts synergistically with the AMPK-BRAF signaling axis.
    RESULTS
    NANOG Is Degraded by SPOP
    NANOG is often expressed in CSCs (Miyazawa et al., 2014; Jeter et al., 2009) and its expression is intimately correlated to poor prognosis in human PCa. Our study indicated that NANOG is a short-lived protein. Treatment with the proteasome inhibitor MG132 dramatically increased the protein level and prolonged the half-life of NANOG (Figures S1A and S1B). Similar results were obtained with the treatment of MLN4924 (Figures S1A and S1B), a potent inhibitor of Cullin-RING ligases by depressing the NEDD8-activating enzyme (NAE) (Ohh et al., 2002), suggest-ing that NANOG is targeted for degradation through a Cullin E3 ubiquitin-ligase-mediated UPS pathway. To search for the Cullin-dependent E3 ligase that targets NANOG for degradation, we screened various dominant-negative forms of Cullins (dnCuls) together with the co-expression of NANOG. Our data showed that the dnCul3, but not other dnCuls, significantly sta-bilized NANOG (Figures 1A and S1C). We further demonstrated that the depletion of Cullin3, but not other Cullins, markedly 
    increased protein level of NANOG in DU145 PCa cells (Figures 1B and 1C). Together, these data indicate that Cullin3 E3 ligase is involved in the control of NANOG degradation in PCa cells.
    To identify the adaptor of Cullin3 that determines NANOG degradation, we analyzed the protein sequence of NANOG and revealed that NANOG contains an evolutionarily conserved SPOP-binding consensus motif (F-p-S-S/T-S/T; F, nonpolar residues; p, polar residues) (Zhuang et al., 2009) using online software (http://elm.eu.org/) (Figure 1D). We therefore examined whether NANOG is a potential substrate of SPOP. To this end, we co-expressed NANOG with SPOP in HEK293T cells and examined the protein level of NANOG. We found that co-expres-sion of SPOP significantly shortened the half-life of NANOG (Fig-ures S1D and S1E). Moreover, both the protein level and the half-life of endogenous NANOG protein were significantly increased upon SPOP depletion in PCa cells and mouse ESCs (mESCs) (Figures 1E, S1F, and S1G). To further confirm this result, we generated two SPOP-deficient DU145 cell lines. Our data showed that the half-life of NANOG is significantly pro-longed in SPOP-deficient DU145 cells (Figure 1F), indicating that NANOG is targeted for degradation by SPOP.
    Next, we tested whether SPOP targets NANOG for ubiquitina-tion. Our data showed that co-expression of SPOP significantly promoted NANOG ubiquitination in HEK293T cells (Figure 1G). In addition, depletion of SPOP markedly decreased NANOG ubiq-uitination in DU145 cells (Figure 1H). Moreover, Cul3-SPOP complex promoted the ubiquitination of NANOG in vitro (Fig-ure S1H). Deletion of the BTB domain in SPOP, which is required for its interaction with Cul3, failed to target NANOG for ubiquiti-nation (Figure S1H), supporting an important role of Cullin3 enzyme activity in SPOP-mediated ubiquitination of NANOG.
    Furthermore, we examined whether NANOG is a binding part-ner of SPOP. Our data indicated that SPOP specifically interacts with NANOG but not other stem cell transcriptional factors, including OCT4, SOX2, and KLF4 (Figure 1I). Notably, the inter-action between endogenous SPOP and NANOG could be readily
    Figure 1. Cullin3/SPOP E3 Ubiquitin Ligase Targets NANOG for Ubiquitination and Degradation
    (A) Hemagglutinin (HA)-NANOG and FLAG-dnCullins were co-expressed in HEK293T cells. Protein levels of NANOG and Cullins were analyzed by western blotting (WB).
    (B) DU145 cells were transfected with control small interfering RNA (siRNA) or Cullins siRNA. The endogenous NANOG and Cullins were analyzed by WB.
    (C) DU145 cells were transfected with control siRNA or three independent Cul3-specific siRNAs. The endogenous NANOG were analyzed by WB.
    (D) Comparison of putative SPOP binding site in NANOG with the SPOP-binding consensus (SBC) motif defined in the known SPOP substrates.