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  • br expressed in of SPOP WT tissues Figure


    expressed in 34.6% of SPOP WT tissues (Figure 7B). As we found that SBC was essential for SPOP-mediated NANOG degradation, we examined whether NANOG is mutated in SBC in those tissues. However, we did not find mutations of NANOG at Ser68 or other sites via Sanger sequencing analysis (data not shown). We therefore examined whether the phosphorylation of NANOG at Ser68 is correlated to the expression level of NANOG in PCa. To this end, we analyzed the phosphorylation of S68 in WT-SPOP PCa tissues. The specificity of NANOG Ser68 phos-phorylation antibody was examined by IHC assay in HEK293T ZVAD FMK expressing NANOG ectopically (Figure S7D). Interestingly, we found that the phosphorylated protein level of NANOG at Ser68 was closely correlated to the total protein level of NANOG in PCa specimens (Figures 7D and 7F). These data support the notion that the stability of NANOG in PCa is controlled at multiple levels (Figures 7G).
    In the current study, we demonstrated that the E3 ubiquitin ligase SPOP governs prostate CSC traits by modulating NANOG stabil-ity. Clinically, cancer-associated mutations in the MATH domain of SPOP disrupt the interaction between SPOP and NANOG, thereby preventing SPOP-mediated destruction of NANOG, stabilizing NANOG from AMPK-BRAF-signaling-axis-induced phosphorylation, and consequently promoting the maintenance of PCa stem-like cells. Our results offer fresh insight into CSC formation and show that SPOP, as a tumor suppressor, pro-motes NANOG ubiquitination and degradation in PCa cells and thus regulates PCa progression.
    AMPK, a master metabolic regulator, has emerged as a poten-tial therapeutic target for various cancer treatments, such as breast cancer and PCa. AMPK activators such as AICAR and metformin have been shown to inhibit prostate cancer cell prolif-eration (Huang et al., 2008; Zakikhani et al., 2008). A direct acti-vator of AMPK inhibits PCa cell growth in the androgen-sensitive and castration-resistant PCa models (Zadra et al., 2014). Metfor-min works together with chemotherapy to block tumor growth and prolong remission via targeting CSCs in breast cancer cell lines (Hirsch et al., 2009). However, the mechanism by which metformin targets CSCs remains unknown. In this study, we found that activation of AMPK attenuated NANOG phosphoryla-tion at Ser68 and thus destabilized NANOG. Thus, our study sug-gests that AMPK activation may function as a novel therapeutic strategy for PCa treatments since it specifically targets at CSCs, thereby offering a very promising way to eliminate the tumor from the root.
    BRAF is an oncogenic protein and its gain-of-function muta-tion has been demonstrated to be tightly correlated with cancer progression in various cancers, such as colon cancer and melanoma (Makrodouli et al., 2011; Lu et al., 2016). Gao’s study showed that activation of B-Raf/Erk pathway can act synergistically to promote androgen independence in the context of the prostate microenvironment in vivo (Gao et al., 2006). However, Raf signaling in prostate CSCs is not well un-derstood yet. In our study, we found that BRAF can phosphor-ylate and stabilize NANOG to promote PCa stem cell-like traits. Although a low frequency of BRAF mutation was reported in PCa, our data showed that regulation of NANOG by BRAF is
    controlled by AMPK-mediated BRAF phosphorylation at Ser729. To further examine the role of Raf in PCa, we analyzed human epidemiological data using the PrognoScan database and found that higher BRAF expression predicted worse pa-tient survival in human PCa (data not shown). Together, our study unveiled a novel mechanism by which BRAF regulates CSC activity via controlling NANOG stability.
    As an essential E3 ubiquitin ligase, the role of SPOP in the maintenance of stem cell properties remains unknown. Our study presents the evidence showing that SPOP controls PCa stemness by degrading NANOG. We also found that overexpres-sion of SPOP in mESCs results in the differentiation of mESCs. Our study discovered an unexplored area in regulating CSC characteristics, which not only contributes to a better under-standing of tumorigenesis but also shed light on a novel strategy of anti-tumor therapeutics targeting at a thorough elimination of the ‘‘seeds,’’ CSCs.
    Detailed methods are provided in the online version of this paper and include the following:
    B Cell Culture
    B In Vivo Xenograft Assay
    B Human Prostate Tumor Tissue and IHC Staining d METHOD DETAILS
    B Alkaline Phosphatase (AP) Staining B In Vitro Phosphorylation Assay
    B Sphere Formation Assay B RNA Interference