br To examine whether the mutation
To examine whether the mutation of SPOP is correlated to aberrant NANOG-mediated gene expression in PCa, we analyzed the gene-expression-based signatures from human clinical specimens with high expression levels of NANOG versus SPOP mutation. By two-class unpaired significance analysis of microarray data obtained from tumor and normal samples with or without SPOP mutation, as well as the patient cases with a high expression of NANOG from TCGA database, we observed an intimate correlation between the enhanced gene expression of mutant SPOP and a high level of NANOG in tumors (fold change > 2; q < 0.05) (Figure S2G). More importantly, our analysis demonstrated that 1,815 genes, including 756 upregulated genes and 1,059 downregulated genes, are affected by SPOP mutations and the high expres-sion level of NANOG, emphasizing the crucial roles of both SPOP and NANOG in cancer progression (Figures S2H and S2I). Indeed, the selected ones from the aforementioned 1,815 genes were influenced by the expression of NANOG (Figures S2J and S2K). Together, these data indicate that SPOP mutation affects the NANOG-regulated gene expression in PCa cells.
Identification of the SPOP-Binding Consensus Motif in NANOG
NANOG contains a conserved putative SPOP binding consensus (SBC) motif 66PDSST70 at its N terminus (Figure 3A). We then examined whether the putative SBC motif is involved in the inter-action between SPOP and NANOG. Our data showed that dele-tion of the putative SBC motif (DPDSST) completely abolished the
binding between NANOG and SPOP in vivo and in vitro (Figures 3B and 3C). Moreover, 3XFLAG of the NANOG SBC motif dramat-ically prolonged its half-life (Figures S3A and S3B) and increased its transcriptional activity (Figure S3C). Importantly, SPOP was unable to promote the ubiquitination and degradation of the SBC-deleted NANOG mutant (Figures 3D and 3F). These data indicate that the SBC motif 66PDSST70 of NANOG is essential for its interaction with SPOP and resultant SPOP-mediated NANOG degradation.
We next explored whether the residues located in the SBC motif involve the interaction between NANOG and SPOP. The simultaneous replacement of Ser68, Ser69, or Thr70 with Ala (3A) in NANOG completely abolished its binding to SPOP (Fig-ure 3B). We also generated a series of NANOG point mutations, including S65A, P66A, D67A, S68A, S69A, T70A, S71A, and P72A and detected their interaction with SPOP. Co-immunopre-cipitation (coIP) assays showed that D67A and S68A, but not other mutants, failed to interact with SPOP (Figure 3G). Consis-tent with these results, SPOP did not promote the degradation of NANOG-D67A and S68A mutants (Figures 3H and 3J). More-over, data from the biotin-labeled peptide pull-down assay showed that SPOP bound to the peptide containing 66PDSST70 but not the mutated 66PDAST70 motif of NANOG (Figure 3K). These data together indicate that the SBC motif of NANOG, especially D67 and S68 residues, are essential for SPOP-medi-ated NANOG degradation.
Cancer-Associated NANOG Mutation within the SBC Motif Is Resistant to SPOP-Mediated Degradation
Our data showed that the SBC motif is essential for NANOG stability, we thus analyzed whether there is any mutation at the SBC motif of NANOG in cancers. Interestingly, a Ser68Tyr (S68Y) mutation in the SBC motif of NANOG was identified in endometrium carcinoma tissues by analyzing TCGA and COSMIC databases (https://cancer.sanger.ac.uk/cosmic/) (Fig-ure 4A). Through computational modeling analyses, we found that the amino acid sequence 66PDSST70 of NANOG binds to an extended groove in the MATH domain of SPOP (Figure S4A). However, the cancer-associated mutation 66PDYST70 in the NANOG mutant increased steric hindrance and impaired its interaction with SPOP (Figures S4B), suggesting that the can-cer-associated mutant S68Y may affect the interaction between NANOG and SPOP.
To confirm this result, we examined the interaction between NANOG S68Y and SPOP. Our data showed that the NANOG S68Y mutant almost completely lost the ability to interact with SPOP (Figure 4B). Moreover, SPOP bound to the peptide motif of WT-NANOG containing 66PDSST70, but not the point-mutated one within the sequence of 66PDYST70 (Figure 4C). Furthermore, SPOP promoted the degradation of WT-NANOG but not NANOG-S68Y (Figures 4D–4F). Consistently, SPOP promoted the ubiquitination of NANOG-WT but not NANOG-S68Y (Fig-ure 4G). Also, the NANOG S68Y mutant exhibited an increased transcriptional activity compared with that of WT-NANOG (Figure S4C).
Next, we examined whether NANOG-S68Y mutant regulates the function of CSCs. Sphere formation assays indicated that overexpression of NANOG-WT or NANOG S68Y significantly increased the efficacy of sphere formation (Figures 4H and 4I).