Prostate cancer-associated mutations in speckle-type POZ protein (SPOP) regulate steroid receptor coactivator 3 protein turnover.
ABSTRACT: The p160 steroid receptor coactivators (SRCs) SRC-1, SRC-2 [nuclear receptor coactivator (NCOA)2], and SRC-3 [amplified in breast cancer 1 (AIB1)/NCOA3] are key pleiotropic "master regulators" of transcription factor activity necessary for cancer cell proliferation, survival, metabolism, and metastasis. SRC overexpression and overactivation occur in numerous human cancers and are associated with poor clinical outcomes and resistance to therapy. In prostate cancer (PC), the p160 SRCs play critical roles in androgen receptor transcriptional activity, cell proliferation, and resistance to androgen deprivation therapy. We recently demonstrated that the E3 ubiquitin ligase adaptor speckle-type poxvirus and zinc finger (POZ) domain protein (SPOP) interacts directly with SRC-3 and promotes its cullin 3-dependent ubiquitination and proteolysis in breast cancer, thus functioning as a potential tumor suppressor. Interestingly, somatic heterozygous missense mutations in the SPOP substrate-binding cleft recently were identified in up to 15% of human PCs (making SPOP the gene most commonly affected by nonsynonymous point mutations in PC), but their contribution to PC pathophysiology remains unknown. We now report that PC-associated SPOP mutants cannot interact with SRC-3 protein or promote its ubiquitination and degradation. Our data suggest that wild-type SPOP plays a critical tumor suppressor role in PC cells, promoting the turnover of SRC-3 protein and suppressing androgen receptor transcriptional activity. This tumor suppressor effect is abrogated by the PC-associated SPOP mutations. These studies provide a possible explanation for the role of SPOP mutations in PC, and highlight the potential of SRC-3 as a therapeutic target in PC.
Project description:Somatic missense mutations in the substrate-binding pocket of the E3 ubiquitin ligase adaptor SPOP are present in up to 15% of human prostate adenocarcinomas, but are rare in other malignancies, suggesting a prostate-specific mechanism of action. SPOP promotes ubiquitination and degradation of several protein substrates, including the androgen receptor (AR) coactivator SRC-3. However, the relative contributions that SPOP substrates may make to the pathophysiology of SPOP-mutant (mt) prostate adenocarcinomas are unknown. Using an unbiased bioinformatics approach, we determined that the gene expression profile of prostate adenocarcinoma cells engineered to express mt-SPOP overlaps greatly with the gene signature of both SRC-3 and AR transcriptional output, with a stronger similarity to AR than SRC-3. This finding suggests that in addition to its SRC-3-mediated effects, SPOP also exerts SRC-3-independent effects that are AR-mediated. Indeed, we found that wild-type (wt) but not prostate adenocarcinoma-associated mutants of SPOP promoted AR ubiquitination and degradation, acting directly through a SPOP-binding motif in the hinge region of AR. In support of these results, tumor xenografts composed of prostate adenocarcinoma cells expressing mt-SPOP exhibited higher AR protein levels and grew faster than tumors composed of prostate adenocarcinoma cells expressing wt-SPOP. Furthermore, genetic ablation of SPOP was sufficient to increase AR protein levels in mouse prostate. Examination of public human prostate adenocarcinoma datasets confirmed a strong link between transcriptomic profiles of mt-SPOP and AR. Overall, our studies highlight the AR axis as the key transcriptional output of SPOP in prostate adenocarcinoma and provide an explanation for the prostate-specific tumor suppressor role of wt-SPOP.
Project description:Steroid receptor co-activator-3 (SRC-3/AIB1) is an oncogene that is amplified and overexpressed in many human cancers. However, the molecular mechanisms that regulate 'activated SRC-3 oncoprotein' turnover during tumorigenesis remain to be elucidated. Here, we report that speckle-type POZ protein (SPOP), a cullin 3 (CUL3)-based ubiquitin ligase, is responsible for SRC-3 ubiquitination and proteolysis. SPOP interacts directly with an SRC-3 phospho-degron in a phosphorylation-dependent manner. Casein kinase I? phosphorylates the S102 in this degron and promotes SPOP-dependent turnover of SRC-3. Short hairpin RNA knockdown and overexpression experiments substantiated that the SPOP/CUL3/Rbx1 ubiquitin ligase complex promotes SRC-3 turnover. A systematic analysis of the SPOP genomic locus revealed that a high percentage of genomic loss or loss of heterozygosity occurs at this locus in breast cancers. Furthermore, we demonstrate that restoration of SPOP expression inhibited SRC-3-mediated oncogenic signaling and tumorigenesis, thus positioning SPOP as a tumor suppressor.
Project description:Androgen receptor (AR) signaling is a key driver of prostate cancer (PC). While androgen-deprivation therapy is transiently effective in advanced disease, tumors often progress to a lethal castration-resistant state (CRPC). We show that recurrent PC-driver mutations in speckle-type POZ protein (SPOP) stabilize the TRIM24 protein, which promotes proliferation under low androgen conditions. TRIM24 augments AR signaling, and AR and TRIM24 co-activated genes are significantly upregulated in CRPC. Expression of TRIM24 protein increases from primary PC to CRPC, and both TRIM24 protein levels and the AR/TRIM24 gene signature predict disease recurrence. Analyses in CRPC cells reveal that the TRIM24 bromodomain and the AR-interacting motif are essential to support proliferation. These data provide a rationale for therapeutic TRIM24 targeting in SPOP mutant and CRPC patients.
Project description:The three members of the p160 family of steroid receptor coactivators (SRC-1, SRC-2, and SRC-3) steer the functional output of numerous genetic programs and serve as pleiotropic rheostats for diverse physiological processes. Since their discovery ?15 years ago, the extraordinary sum of examination of SRC function has shaped the foundation of our knowledge for the now 350+ coregulators that have been identified to date. In this perspective, we retrace our steps into the field of coregulators and provide a summary of selected seminal work that helped define the SRCs as masters of systems biology.
Project description:The E3 ubiquitin ligase adaptor speckle-type POZ protein (SPOP) is frequently dysregulated in prostate adenocarcinoma (PC), via either somatic mutations or mRNA downregulation, suggesting an important tumour suppressor function. To examine its physiologic role in the prostate epithelium in vivo, we 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, our data highlight SPOP as an important regulator of luminal epithelial cell proliferation and c-MYC expression in prostate physiology, identify c-MYC as a novel bona fide SPOP substrate, and help explain the frequent inactivation of SPOP in human PC. We propose SPOPMT-induced stabilization of c-MYC protein as a novel mechanism that can increase total c-MYC levels in PC cells, in addition to amplification of c-MYC locus.
Project description:Geminin and its binding partner Cdt1 are essential for the regulation of DNA replication. Here we show that the CULLIN3 E3 ubiquitin ligase adaptor protein SPOP binds Geminin at endogenous level and regulates DNA replication. SPOP promotes K27-linked non-degradative poly-ubiquitination of Geminin at lysine residues 100 and 127. This poly-ubiquitination of Geminin prevents DNA replication over-firing by indirectly blocking the association of Cdt1 with the MCM protein complex, an interaction required for DNA unwinding and replication. SPOP is frequently mutated in certain human cancer types and implicated in tumorigenesis. We show that cancer-associated SPOP mutations impair Geminin K27-linked poly-ubiquitination and induce replication origin over-firing and re-replication. The replication stress caused by SPOP mutations triggers replication catastrophe and cell death upon ATR inhibition. Our results reveal a tumor suppressor role of SPOP in preventing DNA replication over-firing and genome instability and suggest that SPOP-mutated tumors may be susceptible to ATR inhibitor therapy.
Project description:Gene activation by steroid hormone receptors involves the recruitment of the steroid receptor coactivator (SRC)/p160 coactivator LXXLL motifs to activation function 2 (AF2) in the ligand binding domain. For the androgen receptor (AR), AF2 also serves as the interaction site for the AR NH(2)-terminal FXXLF motif in the androgen-dependent NH(2)-terminal and carboxyl-terminal (N/C) interaction. The relative importance of the AR AF2 site has been unclear, since the AR FXXLF motif interferes with coactivator recruitment by competitive inhibition of LXXLL motif binding. In this report, we identified the X chromosome-linked melanoma antigen gene product MAGE-11 as an AR coregulator that specifically binds the AR NH(2)-terminal FXXLF motif. Binding of MAGE-11 to the AR FXXLF alpha-helical region stabilizes the ligand-free AR and, in the presence of an agonist, increases exposure of AF2 to the recruitment and activation by the SRC/p160 coactivators. Intracellular association between AR and MAGE-11 is supported by their coimmunoprecipitation and colocalization in the absence and presence of hormone and by competitive inhibition of the N/C interaction. AR transactivation increases in response to MAGE-11 and the SRC/p160 coactivators through mechanisms that include but are not limited to the AF2 site. MAGE-11 is expressed in androgen-dependent tissues and in prostate cancer cell lines. The results suggest MAGE-11 is a unique AR coregulator that increases AR activity by modulating the AR interdomain interaction.
Project description:MicroRNAs are important epigenetic regulators of protein expression by triggering degradation of target mRNAs and/or inhibiting their translation. Dysregulation of microRNA expression has been reported in several cancers, including prostate cancer (PC). We comprehensively characterized the proteomic footprint of a panel of 12 microRNAs that are potently suppressed in metastatic PC (SiM-miRNAs: miR-1, miR-133a, miR-133b, miR-135a, miR-143-3p, miR-145-3p, miR-205, miR-221-3p, miR-221-5p, miR-222-3p, miR-24-1-5p, and miR-31) using reverse-phase proteomic arrays. Re-expression of these SiM-miRNAs in PC cells suppressed cell proliferation and targeted key oncogenic pathways, including cell cycle, apoptosis, Akt/mammalian target of rapamycin signaling, metastasis and the androgen receptor (AR) axis. However, only 12%, at most, of these observed protein expression changes could be explained by predicted direct binding of miRNAs to corresponding mRNAs, suggesting that the majority of these proteomic effects result indirectly. AR and its steroid receptor coactivators (SRCs; SRC-1, -2 and -3) were recurrently affected by these SiM-miRNAs. In agreement, we identified inverse correlations between expression of these SiM-miRNAs and early clinical recurrence, as well as with AR transcriptional activity in human PC tissues. We also identified robust induction of miR-135a by androgen and strong direct binding of AR to the miR-135a locus. As miR-135a potently suppresses AR expression, this results in a negative feedback loop that suppresses AR protein expression in an androgen-dependent manner, while de-repressing AR expression upon androgen deprivation. Our results demonstrate that epigenetic silencing of these SiM-miRNAs can result in increased AR axis activity and cell proliferation, thus contributing to disease progression. We further demonstrate that a negative feedback loop involving miR-135a can restore AR expression under androgen-deprivation conditions, thus contributing to the upregulation of AR protein expression in castration-resistant PC. Finally, our unbiased proteomic profiling demonstrates that the majority of actual protein expression changes induced by SiM-miRNAs cannot be explained based on predicted direct interactions.
Project description:The p160/Steroid Receptor Coactivators SRC-1, SRC-2/GRIP1, and SRC-3/AIB1 are important regulators of Estrogen Receptor alpha (ER?) activity. However, whereas the functions of SRC-1 and SRC-3 in breast tumourigenesis have been extensively studied, little is known about the role of SRC-2. Previously, we reported that activation of the cAMP-dependent protein kinase, PKA, facilitates ubiquitination and proteasomal degradation of SRC-2 which in turn leads to inhibition of SRC-2-coactivation of ER? and changed expression of the ER? target gene, pS2. Here we have characterized the global program of transcription in SRC-2-depleted MCF-7 breast cancer cells using short-hairpin RNA technology, and in MCF-7 cells exposed to PKA activating agents. In order to identify genes that may be regulated through PKA-induced downregulation of SRC-2, overlapping transcriptional targets in response to the respective treatments were characterized. Interestingly, we observed decreased expression of several breast cancer tumour suppressor genes (e.g., TAGLN, EGR1, BCL11b, CAV1) in response to both SRC-2 knockdown and PKA activation, whereas the expression of a number of other genes implicated in cancer progression (e.g., RET, BCAS1, TFF3, CXCR4, ADM) was increased. In line with this, knockdown of SRC-2 also stimulated proliferation of MCF-7 cells. Together, these results suggest that SRC-2 may have an antiproliferative function in breast cancer cells.
Project description:SPOP, an adaptor protein for E3 ubiquitin ligase can function as a tumor-suppressor or a tumor-enhancer. In castration-resistant prostate cancer (CRPC), it inhibits tumorigenesis by degrading many oncogenic targets, including androgen receptor (AR). Expectedly, SPOP is the most commonly mutated gene in CRPC (15%), which closely correlates with poor prognosis. Importantly, 85% of tumors that retain wild-type SPOP show reduced protein levels, indicating that SPOP downregulation is an essential step in CRPC progression. However, the underlying molecular mechanism remains unknown. This study uncovered the first mechanism of SPOP regulation in any type of cancer. We identified SPOP as a direct substrate of Aurora A (AURKA) using an innovative technique. AURKA directly phosphorylates SPOP at three sites, causing its ubiquitylation. SPOP degradation drives highly aggressive oncogenic phenotypes in cells and in vivo including stabilizing AR, ARv7 and c-Myc. Further, SPOP degrades AURKA via a feedback loop. SPOP upregulation is one of the mechanisms by which enzalutamide exerts its efficacy. Consequently, phospho-resistant SPOP fully abrogates tumorigenesis and EMT in vivo, and renders CRPC cells sensitive to enzalutamide. While genomic mutations of SPOP can be treated with gene therapy, identification of AURKA as an upstream regulator of SPOP provides a powerful opportunity for retaining WT-SPOP in a vast majority of CRPC patients using AURKA inhibitors ± enzalutamide, thereby treating the disease and inhibiting its progression.