br E PC cells were transfected with the
(E) PC3 cells were transfected with the indicated siRNA. The endogenous NANOG were analyzed by WB.
(F) DU145 SPOP wild-type (WT) or KO cell lines were constructed by the lenti-CRISPR system; cells were treated with cycloheximide (CHX,10 mg/mL) for indicated times. Protein levels of NANOG and SPOP were analyzed by WB.
(G) FLAG-NANOG, HA-SPOP, and His-ubiquitin were co-expressed in HEK293T cells. After treatment with MG132 (10 mM) for 6 hr, the nickel-nitrilotriacetic 50-07-7 (Ni-NTA) ubiquitination assay was performed and analyzed by WB.
(H) DU145 cells were transfected with the indicated siRNA and constructs for 3 days, the cells were treated with MG132 (5 mM) for 12 hr, cell lysate was immunoprecipitated (IP) with anti-NANOG antibody, and the ubiquitination of NANOG was detected by WB using the anti-His antibody.
(I) FLAG-SPOP and indicated HA-tag plasmids were co-expressed in HEK293T cells. Cell lysates were prepared for coIP and WB. Cells were treated with MG132 (10 mM) for 6 hr before harvesting.
(J) DU145 cell lysates were prepared for coIP with SPOP antibody and WB. Cells were treated with MG132 (10 mM) for 6 hr before harvesting.
(K) E14 mESCs cell lysates were prepared for coIP with SPOP antibody and WB. Cells were treated with MG132 (10 mM) for 6 hr before harvesting.
(L) Representative sphere images from each condition of DU145 cells. Scale bar, 200 mm.
(M) Indicated retrovirus infected DU145 tumor spheres were dissociated, and equal numbers of cells were passaged for three generations. Spheres counts are normalized to the first-generation scrambled short hairpin RNA (shRNA) spheres. Data are means ± SEM (n = 3). **p < 0.01 versus control shRNA (shCon) (Student’s t test).
(N and O) Growth of DU145 cell- (N) and 22RV1 cell- (O) derived tumors in nude mice infected with retrovirus expressing indicated shRNAs of three different cell densities. Frequency of CSCs is estimated as per extreme limiting dilution analysis (ELDA) calculating website, and p value represents overall test for differences in stem cell frequencies between the two groups; 1/1 in SPOP shRNA tumors means stem cell frequency (100%); *p < 0.05, ***p < 0.001 versus negative control (NC).
(legend on next page)
detected in PCa cells (Figures 1J and S1I) and E14 mESCs (Fig-ure 1K). Moreover, our data showed that NANOG specifically interacts with SPOP but not other Cullin3-based E3 ligase adaptor proteins (Figure S1J). These results together indicate that SPOP is the adaptor responsible for interacting with NANOG and targeting it for ubiquitination by Cullin3 E3 ubiquitin ligase complex.
SPOP Represses Stem-Cell-like Properties via Its Regulation of NANOG Stability
NANOG expression in PCa is closely correlated to CSC charac-teristics, we thus examined whether SPOP can regulate CSC-like properties by targeting NANOG for degradation. To this end, we examined the effect of SPOP on the oncosphere-forming capacity of PCa cells, which is a standard for the measurement of CSC functions (Zhang et al., 2010). The number of oncospheres formed over multiple passages represents the self-renewal activ-ity, while the size of sphere indicates cell proliferation capability (Dontu et al., 2004). Our data showed that compared to control cells infected with empty vector, both AR-negative DU145 and AR-positive 22RV1 cells lacking SPOP had a progressive in-crease in self-renewal capacity and the ability to form daughter spheres. Notably, simultaneous depletion of NANOG abolished the phenotype induced by SPOP depletion (Figures 1L, 1M, S1L, and S1M). Moreover, SPOP knockout (KO) DU145 cells also showed an increased capability of forming oncospheres (Figure S1K).
We further investigated whether SPOP can affect the tumor progression via its mediation of NANOG degradation using a nude mouse xenograft model. Our data showed that depletion of SPOP in AR-negative DU145 and AR-positive 22RV1 PCa cells significantly promoted tumor growth, whereas simulta-neous depletion of NANOG abolished such phenotype induced by SPOP depletion (Figures S1N and S1O). Furthermore,
SPOP-depleted oncospheres developed more tumors, while simultaneous knockdown of NANOG abolished the phenotype derived from SPOP depletion (Figures 1N and 1O). Moreover, our data showed that knockdown of NANOG greatly reduced the ability of tumor formation when examined by the injection of as few as 1,000 oncospheres into nude mice (Figures 1N, 1O, S1N, and S1O).
To further examine the role of SPOP in CSC function, we per-formed a clonal serial in vivo repopulation assay, which is a widely accepted functional assay to examine the self-renewal capacity of maintaining the long-term clonal growth of CSCs (Kreso and Dick, 2014). Our data from triplicate experiments of serial transplantation showed that depletion of SPOP in both AR-negative DU145 and AR-positive 22RV1 PCa cells elevated the in vivo cell renewal efficacy in a NANOG-dependent manner (Figures 1N, 1O, S1N, and S1O). These data together indicated that SPOP negatively regulates PCa stem cells maintenance in a NANOG-dependent manner and confirmed that regulation of PCa stem cells properties by SPOP-mediated NANOG degrada-tion is independent of AR.