Role of SPOP in Prostate Cancer

Speckle-Type POZ Protein (SPOP) Mutations Regulate the Steroid Receptor Coactivator-3/Androgen Receptor Axis: A New Pathogenetic Mechanism and Therapeutic Target in Prostate Cancer

Recent exome sequencing studies have identified the E3 ubiquitin ligase adaptor speckle-type POZ protein (SPOP) as the gene most commonly affected by somatic non-synonymous point mutations in prostate cancer. However, the role of these SPOP mutants in prostate cancer pathophysiology was unknown.

SPOP interacts directly with SRC-3 and promotes its cullin 3 (Cul3)-dependent ubiquitination and proteolysis in breast cancer (Fig. 1), thus functioning as a tumor suppressor (Geng et al. PNAS 2013). SPOP contains two conserved domains: an N-terminal MATH (Meprin and Traf Homology) domain that recruits substrate proteins, and a C-terminal BTB (Bric-a-brac/Tramtrack/Broad complex) domain that interacts with Cul3. All SPOP mutations reported in prostate cancer affect conserved residues in the structurally defined substrate-binding pocket (Fig. 1), raising the hypothesis that they can modify substrate specificity. SPOP mutations comprise an early event in prostate carcinogenesis.

We have now discovered that prostate cancer-associated SPOP mutants cannot interact with SRC-3 protein or promote its degradation (Fig. 1), thus providing a possible explanation of the impact of SPOP mutations in prostate cancer (Geng et al. PNAS 2013). As SRC-3 can potently promote AR transcriptional activity and pleiotropic oncogenic signaling necessary for cancer cell proliferation, survival, metabolism and metastasis, we hypothesized that SRC-3 could be one of the major SPOP substrates mediating the effect of mutant SPOP in prostate cancer and investigated the impact of wild-type and mutant SPOP on SRC expression and function. Overexpression of SPOP-WT potently promoted the degradation of SRC-3 protein, but not SRC-1 or SRC-2 protein. All prostate cancer-associated SPOP mutants tested failed to promote SRC-3 ubiquitination and protein degradation. We further documented the physical interaction of SPOP-WT with SRC-3 (but not SRC-1 or SRC-2), and this interaction was abolished in these prostate cancer-associated SPOP mutants (Geng et al. PNAS 2013). In prostate cancer cells, SPOP-WT suppresses SRC-3 protein expression, cell proliferation and AR transcriptional activity, while this effect is abolished or significantly attenuated by the prostate cancer-associated SPOP mutations.

Using an unbiased bioinformatics approach, we determined that the gene expression profile of PC cells engineered to express mutant-SPOP (mt-SPOP) overlaps greatly with the gene signature of both SRC3 and AR transcriptional output, but to a stronger degree for AR than SRC3. This finding suggests that, in addition to SRC3-mediated effects, SPOP also exerts SRC3-independent/AR-mediated functions (Fig. 2 and Geng et al). Indeed, we found that wild-type (wt)-SPOP, but not the PC-associated mt-SPOPs, can promote ubiquitination and degradation of AR itself, by interacting with a SPOP-binding motif in the hinge region of AR (Fig. 3-4 and Geng et al). Xenografts of PC cells expressing mt-SPOP expressed more AR protein and grew faster in immunocompromised mice than those expressing wt-SPOP. Genetic ablation of SPOP resulted in increased AR protein levels in the mouse prostate. Examination of several publicly available human PC datasets confirmed the strong link between the transcriptomic profiles of mt-SPOPs and AR. Thus, our studies suggested the AR axis as the key transcriptional output of SPOP in PC and provide an explanation for the prostate-specific tumor suppressor role of wt-SPOP.

Our findings were subsequently confirmed by the Cancer Genome Atlas (TCGA) comprehensive molecular analysis of 333 primary prostate carcinomas, which showed that SPOP-mutant tumors have the highest levels of AR-induced transcripts.

Figure 1

Our data suggest that wild-type SPOP plays a critical tumor suppressor role in prostate cancer cells, promoting the turnover of SRC-3 protein and suppressing AR transcriptional activity (Fig 1A). This tumor suppressor effect is abrogated by the prostate cancer-associated SPOP mutations (Fig 1B) (Geng et al. PNAS 2013). These studies provide a possible explanation for the role of SPOP mutations in prostate cancer, and highlight the potential of SRC-3 as a therapeutic target in prostate cancer.

Figure 2

Global gene expression profiling identifies the AR transcriptional output as the top enriched gene set in PC cells expressing mutant SPOPs.

A. Hierarchical clustering of gene expression profiles of Abl PC cells transfected with control vector, wt-SPOP or the SPOP mutants F102C, F133V, F133L (genes differentially expressed, t-test p<0.05, fold change exceeding 4/3x) demonstrates that all three SPOP mutants have highly similar effects on the PC transcriptome, which are very distinct (essentially inverted) from the effects of the wt-SPOP.

B. The top Molecular Signature Database (MSigDB) match for the mt-SPOP gene signature, out of 10,295 available gene sets analyzed in an unbiased fashion, is the “NELSON_RESPOSE_TO_ANDROGEN_UP”. Also, three out of the top five chemical and genetic perturbation (CGP) gene sets in PC correspond to androgen-induced transcriptomic responses.

C. The AR Activity Score was calculated, based on a previously published gene signature of androgen-stimulated LNCaP cells, for Abl cells expressing control vector, wt-SPOP, or mt-SPOP. Compared to control vector, cells expressing wt-SPOP exhibit a lower AR Activity Score, while mt-SPOPs increase the AR Activity Score.

D. Gene Set Enrichment Analysis (GSEA) revealed that SRC3-upregulated genes (genes down-regulated by SRC-3 siRNA) are significantly enriched in the mt-SPOP gene signatures. The values on the y axis for each graph represent enrichment scores (corresponding to the magnitude of the enrichments for each analysis). For each graph, the Normalized Enrichment Score (NES, computed via the GSEA analysis) and the significance of the enrichment (q = false discovery rate also computed via the GSEA analysis). The NES scores range from 1.35 to 1.47 (all q<0.02). However, we found that the mt-SPOP gene signatures show stronger enrichment, with NES ranging from 1.88 to 2.71 (q<0.001), for androgen-induced genes (genes down-regulated by AR siRNA or induced by androgen treatment of PC cells).

Figure 3

Ectopic overexpression of wt-SPOP post-translationally regulates AR in PC cells.

Abl PC cells engineered to express, under a tetracycline-inducible promoter, SPOP-wt or its PC-associated mutants, were treated with 0, 50 or 500 ng/ml of doxycycline (Dox) for 24 hours. Following this, cells were collected and cell lysates were prepared. Immunoblot analyses were conducted for the expression of AR, SPOP and β-Actin in the cell lysates. SPOP-wt, but not its PC-associated mutants, significantly suppressed AR protein expression in Abl PC cells. The numbers beneath the bands represent densitometry analysis performed on representative blots from each cell line.

Wt-SPOP promotes ubiquitination of AR Protein in vitro and this activity is abrogated by the PC-associated SPOP Mutations. 293T cells were co-transfected with pcDNA3-HA-human Ubiquitin (1 μg) and pcDNA3-AR-Flag (1 μg), together with same amount (1 μg) of pcDNA3.1 expression vectors for Wt-SPOP or its PC-associated mutated variants (SPOP-F102C, SPOP-F133V, SPOP-F125V, SPOP-S119N, SPOP-Y87C, SPOP-Y87N) or SPOP-C terminal fragment (a.a.172-a.a.374, lacking the MATH domain), respectively. Anti-Flag antibody was used to immunoprecipitate AR protein, and anti-HA-HRP antibody was used to visualize the ubiquitinated AR by immunoblotting. To detect the AR protein (input) in cell lysate samples, anti-AR antibody (Santa Cruz Biotech. Inc) was used. The levels of ubiquinated AR protein were significantly increased when SPOP-wt was also expressed, whereas expression of any PC-associated SPOP mutant effectively inhibited the accumulation of Ub-AR. Furthermore, the SPOP C-terminal fragment (a.a.172-a.a.374) had no effect on AR ubiquitination.

Figure 4

Interaction and binding of SPOP-wt to AR is mediated by a SBC motif (a.a.646-a.a.651) located in the Hinge Region of the AR protein and leads to AR protein degradation. A. Schematic map of AR and its C-terminal truncated variant, ARv7 protein, showing the SBC motif in AR. B. 293T cells were co-transfected with vectors expressing HA-SPOP-wt and Flag-ARwt or -Flag-AR mutants (-Flag-AR-A646D, -Flag-AR-S648N, -Flag-AR-S647F and -Flag-AR-STT648/649/650AAA). The transfected cells were treated with 250 nM of the proteasome inhibitor bortezomib (PS341) for another 8 hours and cells lysates were prepared and utilized for co-IP/immunoblot analysis as in Fig. 2B. Point mutations in the AR SBC motif, specifically A646D, S647F, S648N and STT648/649/650AAA, can abolish the affinity of AR for SPOP-wt.

Deciphering the physiological role of SPOP using in vivo models

To examine the physiologic role of SPOP in the prostate epithelium in vivo, we recently generated mice with prostate-specific biallelic ablation of SPOP. These mice exhibited increased prostate mass, prostate epithelial cell proliferation, and expression of c-MYC protein compared to littermate controls, and eventually developed prostatic intraepithelial neoplasia (PIN). We found that SPOPWT can physically interact with c-MYC protein and, upon exogenous expression in vitro, can promote c-MYC ubiquitination and degradation. This effect was attenuated in PC cells by introducing PC-associated SPOP mutants or upon knockdown of SPOP via short-hairpin-RNA, suggesting that SPOP inactivation directly increases c-MYC protein levels. Gene Set Enrichment Analysis revealed enrichment of Myc-induced genes in transcriptomic signatures associated with SPOPMT. Likewise, we observed strong inverse correlation between c-MYC activity and SPOP mRNA levels in two independent PC patient cohorts. The core SPOPMT; MYCHigh transcriptomic response, defined by the overlap between the SPOPMT and c-MYC transcriptomic programmes, was also associated with inferior clinical outcome in human PCs. Finally, the organoid-forming capacity of SPOP-null murine prostate cells was more sensitive to c-MYC inhibition than that of SPOP-WT cells, suggesting that c-MYC upregulation functionally contributes to the proliferative phenotype of SPOP knock-out prostates. Taken together, these data highlight SPOP as an important regulator of luminal epithelial cell proliferation and c-MYC expression in prostate physiology, and help explain the frequent inactivation of SPOP in human PC.

References

Geng C, Kaochar S, Li M, Rajapakshe K, Fiskus W, Dong J, Foley C, Dong D,  Zhang L, Kwon OJ, Shah S, Bolaki M, Xin L, Ittmann M, O'Malley BW, Coarfa C, Mitsiades N. SPOP regulates prostate epithelial cell proliferation and promotes ubiquitination and turnover of cMYC oncoprotein. Oncogene 2017 Apr 17. doi: 10.1038/onc.2017.80.

Geng C, Rajapakshe K, Shah SS, Shou J, Eedunuri VK, Foley C, Fiskus W, Rajendran M, Chew SA, Zimmermann M, Bond R, He B, Coarfa C, Mitsiades N. Androgen receptor is the key transcriptional mediator of the tumor suppressor SPOP in prostate cancer Cancer Res. 2014 Oct 1;74(19):5631-43. do: 10.1158/0008-5472.CAN-14-0476. PubMed PMID: 25274033; PubMed Central PMCID: PMC4209379.

Geng C, He B, Xu L, Barbieri CE, Eedunuri VK, Chew SA, Zimmermann M, Bond R, Shou J, Li C, Blattner M, Lonard DM, Demichelis F, Coarfa C, Rubin MA, Zhou P, O'Malley BW, Mitsiades N. Prostate cancer-associated mutations in speckle-type POZ protein (SPOP) regulate steroid receptor coactivator 3 protein turnover. Proc Natl Acad Sci U S A. 2013 Apr 23;110(17):6997-7002.