Speckle-type POZ Protein Mutations Research Articles

Impact of the SPOP Mutant Subtype on the Interpretation of Clinical Parameters in Prostate Cancer.

PurposeMolecular characterization of prostate cancer, including The Cancer Genome Atlas, has revealed distinct subtypes with underlying genomic alterations. One of these core subtypes, SPOP (speckle-type POZ protein) mutant prostate cancer, has previously only been identifiable via DNA sequencing, which has made the impact on prognosis and routinely used risk stratification parameters unclear.MethodsWe have developed a novel gene expression signature, classifier (Subclass Predictor Based on Transcriptional Data), and decision tree to predict the SPOP mutant subclass from RNA gene expression data and classify common prostate cancer molecular subtypes. We then validated and further interrogated the association of prostate cancer molecular subtypes with pathologic and clinical outcomes in retrospective and prospective cohorts of 8,158 patients.ResultsThe subclass predictor based on transcriptional data model showed high sensitivity and specificity in multiple cohorts across both RNA sequencing and microarray gene expression platforms. We predicted approximately 8% to 9% of cases to be SPOP mutant from both retrospective and prospective cohorts. We found that the SPOP mutant subclass was associated with lower frequency of positive margins, extraprostatic extension, and seminal vesicle invasion at prostatectomy; however, SPOP mutant cancers were associated with higher pretreatment serum prostate-specific antigen (PSA). The association between SPOP mutant status and higher PSA level was validated in three independent cohorts. Despite high pretreatment PSA, the SPOP mutant subtype was associated with a favorable prognosis with improved metastasis-free survival, particularly in patients with high-risk preoperative PSA levels.ConclusionUsing a novel gene expression model and a decision tree algorithm to define prostate cancer molecular subclasses, we found that the SPOP mutant subclass is associated with higher preoperative PSA, less adverse pathologic features, and favorable prognosis. These findings suggest a paradigm in which the interpretation of common risk stratification parameters, particularly PSA, may be influenced by the underlying molecular subtype of prostate cancer.

SPOP promotes ATF2 ubiquitination and degradation to suppress prostate cancer progression

BackgroundNext-generation sequencing of the exome and genome of prostate cancers has identified numerous genetic alterations. SPOP (Speckle-type POZ Protein) is one of the most frequently mutated genes in primary prostate cancer, suggesting that SPOP may be a potential driver of prostate cancer. The aim of this work was to investigate how SPOP mutations contribute to prostate cancer development and progression.MethodsTo identify molecular mediators of the tumor suppressive function of SPOP, we performed a yeast two-hybrid screen in a HeLa cDNA library using the full-length SPOP as bait. Immunoprecipitation and Western Blotting were used to analyze the interaction between SPOP and ATF2. Cell migration and invasion were determined by Transwell assays. Immunohistochemistry were used to analyze protein levels in patients’ tumor samples.ResultsHere we identified ATF2 as a bona fide substrate of the SPOP-CUL3-RBX1 E3 ubiquitin ligase complex. SPOP recognizes multiple Ser/Thr (S/T)-rich degrons in ATF2 and triggers ATF2 degradation via the ubiquitin-proteasome pathway. Strikingly, prostate cancer-associated mutants of SPOP are defective in promoting ATF2 degradation in prostate cancer cells and contribute to facilitating prostate cancer cell proliferation, migration and invasion.ConclusionSPOP promotes ATF2 ubiquitination and degradation, and ATF2 is an important mediator of SPOP inactivation-induced cell proliferation, migration and invasion.

Abstract 4448: Mutations in the center MATH domain of Speckle Type BTB/POZ Protein lead to enhanced proteinstability in prostate cancer

Abstract The Speckle type Poz Protein (SPOP) is a tumor suppressor that is often mutated in prostate cancer. Mutation of SPOP is linked to genomic instability and it plays a critical role in prostate cancer initiation. SPOP mutations are found to be in the heterozygous state in patients. The relevance of these mutations on its protein expression has not been systematically investigated. Here we report that mutations in the center MATH domain of SPOP lead to enhanced protein stability in prostate cancer. Among the mutations, F102C, S119N, F125V and WI31G showed increased protein stability. Further we show that these mutations in the MATH domain increase half-life of SPOP protein. Interestingly, the mutant SPOPs enhanced the endogenous wild-type SPOP level. We further explored the structural insights of these SPOP mutants. We present a computational model to predict how SPOP mutations potentially lead to conformational changes within the protein, promoting its stability. Finally, immunohistochemistry in clinical samples of prostate cancer augment our findings. Together, we highlight a subset of clinically relevant SPOP mutations that have impact on endogenous SPOP protein stability, allowing to further understand its role in prostate cancer initiation and evaluating of potentially altered therapeutic response in patients harboring these mutations. Citation Format: Joshua Fried, Vinayak Khattar, Jinlu Ma, Qinghua Zeng, Wei Zhang, Bo Xu. Mutations in the center MATH domain of Speckle Type BTB/POZ Protein lead to enhanced proteinstability in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4448.

TAS3681, a novel type of AR antagonist with AR downregulating activity, as a new targeted therapy for aberrant AR-driven prostate cancer.

298 Background: Recent reports suggest that AR aberrations are involved in the development of metastatic castration-resistant prostate cancer and in resistance to AR-targeted therapies. These aberrations include point mutations, copy number gain, and alternatively spliced AR forms. TAS3681 has demonstrated activity as a pure AR antagonist against several AR mutations and has shown an antitumor effect in an AR-v7 positive xenograft model. In order to further characterize the properties of TAS3681, we investigated its effects in cell-based AR overexpression/stabilization models and assessed the subcellular localization of FL-AR and AR-v7 in enzalutamide (Enz) -resistant cells. Methods: The Nano-bit assay was performed to analyze dimerization of FL-AR and AR-v7 proteins. Prostate cancer (PCa) cells expressing AR-v7 were treated with TAS3681, and nuclear and cytoplasmic fractions were prepared. Then the levels of FL-AR and AR-v7 protein in each fraction were determined by western blotting. AR-overexpressing PCa cells transfected with a wild-type AR expression vector were treated with TAS3681 in the presence of androgen. AR-stabilized PCa cells were transfected with a Speckle-type POZ protein (SPOP) mutant expression vector and then were treated with TAS3681. Results: AR antagonism by TAS3681 caused dose-dependent inhibition of FL-AR homodimerization (HD) and FL-AR/AR-v7 heterodimer formation in the presence of androgen. TAS3681 had no direct inhibitory effect on HD of AR-v7. However, AR downregulation by TAS3681 reduced nuclear AR-v7 protein in Enz-resistant cells under castration conditions. In AR-overexpressing PCa cells, TAS3681 reduced the AR protein and effectively suppressed both AR transactivation and cell proliferation. In contrast to Enz, TAS3681 also effectively reduced the AR protein in AR-stabilized PCa cells harboring SPOP mutation. Conclusions: TAS3681 suppresses aberrant AR activation, including AR overexpression and expression of constitutively active nuclear localized AR-v7, via downregulation of AR/AR-v7. These findings suggest that TAS3681 has the potential to overcome resistance to current AR-targeted therapies.

Functional roles of Speckle-Type Poz (SPOP) Protein in Genomic stability.

Understanding the functional significance of the essential elements in maintaining genomic stability provides insights into the process of tumor initiation and progression, and predicts therapeutic responses. One such element that has recently attracted significant attention is the Speckle-Type Poz Protein (SPOP), an E3 ubiquitin ligase adaptor protein. SPOP is frequently mutated or has altered expression in various cancers, including prostate, renal and endometrial. SPOP is involved in the regulation of proteasome-mediated degradation of several oncoproteins. Moreover, recent data also indicate SPOP's direct involvement in the DNA damage response. SPOP mutants induce alternations in the DNA damage repair pathway by promoting the error-prone Non-homologous end joining (NHEJ) pathway. SPOP has been linked with significant functions in cellular signaling pathways and cancer suppression. This mini-review will discuss recent findings regarding SPOP's role in genomic stability in the pathological setting.

Identification of SPOP related metabolic pathways in prostate cancer.

Speckle-type POZ protein (SPOP), as a cullin-based E3 ubiquitin ligase, has been identified as one of the most frequently mutated genes in prostate cancer (PCa). However, whether SPOP mutations contribute to metabolic reprogramming in PCa remains unknown. Here, integrated studies of transcriptomics and metabolomics as well as lipidomics were performed in matched PCa tumor (PCT) and adjacent non-tumor (ANT) tissues, followed by correlation analysis of SPOP mutations with altered metabolic pathways in SPOP-mutated PCa patients. Interestingly, transcriptomics profiling showed that all SPOP mutations (with 16.7% frequency, 11/66) occurred at the conserved residues in the substrate binding domain of meprin and TRAF homology (MATH). The results of integrated analysis indicated that three metabolic pathways, including tricarboxylic acid (TCA) cycle, fatty acid metabolism and glycerophospholipid metabolism, exhibited obvious upregulation in SPOP-mutated PCT tissues. Furthermore, both correlation analyses based on integrated data and cBioportal revealed that FH, ELOVL2 and ACADL genes might be involved in SPOP-mutation-related upregulation of these metabolic pathways. Taken together, our study provided new insights in understanding the relationship between metabolic pathways and SPOP mutations in PCa.

Intrinsic BET inhibitor resistance in SPOP-mutated prostate cancer is mediated by BET protein stabilization and AKT–mTORC1 activation

Bromodomain and extraterminal domain (BET) protein inhibitors are emerging as promising anti-cancer therapies. The gene encoding the E3 ubiquitin ligase substrate-binding adaptor speckle-type POZ protein (SPOP) is most frequently mutated in prostate cancer. Here we demonstrate that wild-type SPOP binds to and induces ubiquitination and proteasomal degradation of BET proteins (BRD2, BRD3 and BRD4) by recognizing a common degron motif. In contrast, prostate cancer-associated SPOP mutants impair binding and proteasomal degradation of BET proteins, thus inducing their accumulation in prostate cancer cells and patient specimens. Transcriptome and BRD4 cistrome analyses reveal that SPOP mutation enhances BRD4-dependent expression of GTPase RAC1 and cholesterol biosynthesis genes and AKT-mTORC1 activation. SPOP mutant expression confers BET inhibitor resistance and this effect can be overcome by AKT inhibitors. Thus, SPOP mutations promote AKT-mTORC1 activation and intrinsic BET inhibitor resistance by stabilizing BET proteins, suggesting that SPOP mutation can be an effective biomarker to guide BET inhibitor-oriented therapy of prostate cancer.

Abstract 2702: Mutations of SPOP, FOXA1 and IDH1 are associated with prostate cancer metastasis and biochemical outcomes

Abstract Recent genomic studies have shed light on cataloging genomic alterations in prostate cancer. Mechanistic studies allow the use of this information to subclassify the heterogeneous prostate tumors for optimized treatment planning. Speckle-Type POZ Protein (SPOP), Forkhead Box Protein A1 (FOXA1) and Isocitrate Dehydrogenase 1 (IDH1) are among the genes that show frequent point mutations in primary prostate cancers. However, the clinical significance of these gene mutations in cancer patients is less clear. To determine the whether SPOP, FOXA1 and IDH1 mutations are associated with clinical outcomes in prostate cancer, we pyro-sequenced the three genes in prostate cancer patients and evaluated the clinical and prognostic significance. 198 patients from the first hospital of Xi’An Jiaotong University (China) were analyzed. All patients were newly diagnosed with prostate cancer from January 2010 to December 2015. No patient was lost to follow-up. The average follow-up time after the primary diagnosis was 27 months. The Fisher’s exact tests were used to compare categorical data. The risk of metastasis among patients with SPOP, FOXA1 and IDH1 mutations was evaluated using univariate and multivariate logistic regression models. The prognostic significance of clinical and gene mutations was determined using multivariate Cox regression analysis. SPOP mutations were found in 16 patients (8.1%), with all of the mutations clustered in the substrate-binding MATH domain. FOXA1 mutationswere found in nine patients (4.5%), and were clustered in the Forkhead domain. IDH1 mutations were detected in three patients (1.5%). Mutation frequencies of SPOP, FOXA1 and IDH1 were associated with prostate-specific antigen (PSA), a biochemical marker of prostate tumor presence/risk which is decided by Gleason Score, T stage and PSA level, along with prostate cancer PSA failure and metastasis. Among patients with SPOP mutation, 56.3% showed metastasis at the time of diagnosis of primary cancer, compared to only 11.5% of patients with wild-type SPOP. Similarly, the probabilities of FOXA1 and IDH1 mutations with metastasis were approximately 44.4% and 66.7% versus 13.8% and 14.4%, respectively, comparing to their wild-type counterparts. The metastasis risk for patients with SPOP mutation was 1.27 times of the SPOP wild-type patients (P = 0.003). We also found that patients with SPOP (P < 0.0001) and FOXA1 (P = 0.0403) mutations showed higher risk of PSA failure compared with wild-type. The PSA failure risk for patients with SPOP mutation was 35.49 times of the SPOP wild-type patients. In contrast, IDH1 mutation was not associated with PSA failure (P = 0.054). Multivariate analyses demonstrate that SPOP mutation is an independent factor that is associated with prostate cancer metastasis and PSA failure recurrence. These findings underscore the clinical significance for profiling SPOP, FOXA1 and IDH1. Note: This abstract was not presented at the meeting. Citation Format: Jinlu Ma, Suxia Han, Xin Sui, Wenyan Lu, Yonghe Li, Bo Xu. Mutations of SPOP, FOXA1 and IDH1 are associated with prostate cancer metastasis and biochemical outcomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2702. doi:10.1158/1538-7445.AM2017-2702

Inherited determinants of early recurrent somatic mutations in prostate cancer

Prostate cancer is a highly heritable molecularly and clinically heterogeneous disease. To discover germline events involved in prostate cancer predisposition, we develop a computational approach to nominate heritable facilitators of somatic genomic events in the context of the androgen receptor signaling. Here, we use a ranking score and benign prostate transcriptomes to identify a non-coding polymorphic regulatory element at 7p14.3 that associates with DNA repair and hormone-regulated transcript levels and with an early recurrent prostate cancer-specific somatic mutation in the Speckle-Type POZ protein (SPOP) gene. The locus shows allele-specific activity that is concomitantly modulated by androgen receptor and by CCAAT/enhancer-binding protein (C/EBP) beta (CEBPB). Deletion of this locus via CRISPR-Cas9 leads to deregulation of the genes predicted to interact with the 7p14.3 locus by Hi-C chromosome conformation capture data. This study suggests that a polymorphism at 7p14.3 may predispose to SPOP mutant prostate cancer subclass through a hormone-dependent DNA damage response.

Dysregulation of INF2-mediated mitochondrial fission in SPOP-mutated prostate cancer.

Next-generation sequencing of the exome and genome of prostate cancers has identified numerous genetic alternations. SPOP (Speckle-type POZ Protein) was one of the most frequently mutated genes in primary prostate cancer, suggesting SPOP is a potential driver of prostate cancer development and progression. However, how SPOP mutations contribute to prostate cancer pathogenesis remains poorly understood. SPOP acts as an adaptor protein of the CUL3-RBX1 E3 ubiquitin ligase complex that generally recruits substrates for ubiquitination and subsequent degradation. ER-localized isoform of the formin protein inverted formin 2 (INF2) mediates actin polymerization at ER-mitochondria intersections and facilitates DRP1 recruitment to mitochondria, which is a critical step in mitochondrial fission. Here, we revealed that SPOP recognizes a Ser/Thr (S/T)-rich motif in the C-terminal region of INF2 and triggers atypical polyubiquitination of INF2. These ubiquitination modifications do not lead to INF2 instability, but rather reduces INF2 localization in ER and mitochondrially associated DRP1 puncta formation, therefore abrogates its ability to facilitate mitochondrial fission. INF2 mutant escaping from SPOP-mediated ubiquitination is more potent in prompting mitochondrial fission. Moreover, prostate cancer-associated SPOP mutants increase INF2 localization in ER and promote mitochondrial fission, probably through a dominant-negative effect to inhibit endogenous SPOP. Moreover, INF2 is important for SPOP inactivation-induced prostate cancer cell migration and invasion. These findings reveal novel molecular events underlying the regulation of INF2 function and localization, and provided insights in understanding the relationship between SPOP mutations and dysregulation of mitochondrial dynamics in prostate cancer.

SPOP regulates prostate epithelial cell proliferation and promotes ubiquitination and turnover of c-MYC oncoprotein.

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 tumor 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 programs, 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.

Prostate cancer-associated mutation in SPOP impairs its ability to target Cdc20 for poly-ubiquitination and degradation

Recent studies revealed that mutations in SPOP (Speckle-type POZ protein) occur in up to 15% of patients with prostate cancer. However, the physiological role of SPOP in regulating prostate tumorigenesis remains elusive. Here, we identified the Cdc20 oncoprotein as a novel ubiquitin substrate of SPOP. As such, pharmacological inhibition of Cullin-based E3 ligases by MLN4924 could stabilize endogenous Cdc20 in cells. Furthermore, we found that Cullin 3, and, to a less extent, Cullin 1, specifically interacted with Cdc20. Depletion of Cullin 3, but not Cullin 1, could upregulate the abudance of Cdc20 largely via prolonging Cdc20 half-life. Moreover, SPOP, the adaptor protein of Cullin 3 family E3 ligase, specifically interacted with Cdc20, and promoted the poly-ubiquitination and subsequent degradation of Cdc20 in a degron-dependent manner. Importantly, prostate cancer-derived SPOP mutants failed to interact with Cdc20 to promote its degradation. As a result, SPOP-deficient prostate cancer cells with elevated Cdc20 expression became resistant to a pharmacological Cdc20 inhibitor. Therefore, our results revealed a novel role of SPOP in tumorigenesis in part by promoting the degradation of the Cdc20 oncoprotein.

Abstract 2752: Characterization of the ATM-SPOP pathway in prostate cancer tumorigenesis

Abstract The Speckle type Poz Protein (SPOP), an E3 ubiquitin ligase adaptor, has recently been identified as the gene that has the most common somatic point mutations in prostate cancer. SPOP mutations are associated with genomic alterations, indicating a role for SPOP in the maintenance of genome stability. We, and others, have recently demonstrated a critical role of SPOP in the DNA damage response (DDR), suggesting SPOP mutants may represent a subgroup of patients that have hyper sensitivity to DNA damaging therapies. However, how SPOP mutations might impact its function and their roles in the progress of prostate tumorigenesis remain to be extensively studied. Genomic studies have found that SPOP mutations are clustered within its substrate binding pocket. Of a particular interest, Serine119 is found to be frequently mutated to Asparagine (S119N). Using computational modeling, we found that Ser119 resides in the SBC-MATH binding interface and is in close contact with the non-polar residue of the SPOP-binding consensus motif. Therefore we hypothesized that phosphorylation of Serine 119 might directly affect substrate binding. First we characterized prostate cancer cells expressing this mutation and we found that they displayed a suboptimal DNA damage response with prolonged DNA repair. Using an in situ proximity ligation assay, we demonstrate that Serine 119 is essential for SPOP ionizing irradiation induced interaction with the Ataxia Telangiectasia Mutated kinase (ATM), a major hub protein in the DDR. With the evidence of SPOP being a phospho-protein, we further provide in vitro evidence that ATM phosphorylates SPOP on Serine 119 in response to DNA damage. Characterization of the functional significance of ATM-mediated SPOP phosphorylation indicates a wide range of downstream targets regulating cell cycle progression and DNA repair. Taken together, our data reveal a critical pathway linking ATM and SPOP in regulation of prostate cancer initiation and therapeutic responses to DNA damage. This also provides the first evidence of a pathophysiological relevant mutation linked to ATM phosphorylation in the DDR. Citation Format: Joshua Fried, Rebecca Boohaker, Qinghua Zeng, Wei Zhang, Bo Xu. Characterization of the ATM-SPOP pathway in prostate cancer tumorigenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2752.

Abstract 62: Functional analysis of endometrial cancer mutants of the E3 ubiquitin ligase adaptor protein, SPOP

Abstract The purpose of this study is to functionally annotate endometrial cancer (EC) specific mutants of SPOP (Speckle-type POZ protein). SPOP is the substrate adaptor for the E3 ligase complex CUL3-SPOP-RBX1, which is responsible for mediating the ubiquitination, and subsequent proteasomal degradation of a number of protein substrates including the SRC3 (NCOA3/AIB1) oncoprotein. Our laboratory has previously shown that SPOP is frequently mutated in serous and clear cell ECs, two clinically aggressive subtypes of EC. Importantly, the vast majority of SPOP mutants localized within the MATH domain, which is responsible for substrate binding. We hypothesize that EC associated SPOP mutations encode loss-of-function mutants that abolish or attenuate substrate degradation. To test this hypothesis, we initially focused our attention on five SPOP mutations we uncovered in ECs (E47K, S80R, P94A, M117I and R121Q). HEK293 cells were transiently co-transfected with a HA-tagged SRC3-expressing construct and a MYC-tagged construct expressing either vector alone, wildtype SPOP, or mutant forms of SPOP. Protein lysates from transfected cells were subjected to Western blotting to measure exogenous levels of HA-SRC3 and MYC-SPOP. Cells exogenously expressing wildtype SPOP or the E47K, P94A, M117I or R121Q mutants exhibited lower levels of HA-SRC3 than cells expressing the vector control. In contrast, cells expressing the S80R mutant had elevated levels of HA-SRC3 compared to cells expressing either wildtype SPOP or the vector control. Co-immumoprecipitation experiments are being performed to assess the substrate binding capacity of SPOP mutants. Our findings indicate that the SPOP-S80R mutation, which is recurrent in EC, has an impaired ability to down-regulate SRC3 protein levels compared to wildtype SPOP. Ongoing and future studies aim to elucidate the mechanism that underlies the effect of SPOP-S80R, and to functionally annotate additional SPOP mutations in EC. Citation Format: Fred Lozy, Mary Ellen Urick, Meghan Rudd, Deena Maurer, Daphne W. Bell. Functional analysis of endometrial cancer mutants of the E3 ubiquitin ligase adaptor protein, SPOP. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 62.

TRIM24 Is an Oncogenic Transcriptional Activator in Prostate Cancer

Androgen receptor (AR) signaling is a key driver of prostate cancer (PC). While androgen-deprivation therapyis 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 ARsignaling, 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.

Higher-order oligomerization promotes localization of SPOP to liquid nuclear speckles.

Membrane‐less organelles in cells are large, dynamic protein/protein or protein/RNA assemblies that have been reported in some cases to have liquid droplet properties. However, the molecular interactions underlying the recruitment of components are not well understood. Herein, we study how the ability to form higher‐order assemblies influences the recruitment of the speckle‐type POZ protein (SPOP) to nuclear speckles. SPOP, a cullin‐3‐RING ubiquitin ligase (CRL3) substrate adaptor, self‐associates into higher‐order oligomers; that is, the number of monomers in an oligomer is broadly distributed and can be large. While wild‐type SPOP localizes to liquid nuclear speckles, self‐association‐deficient SPOP mutants have a diffuse distribution in the nucleus. SPOP oligomerizes through its BTB and BACK domains. We show that BTB‐mediated SPOP dimers form linear oligomers via BACK domain dimerization, and we determine the concentration‐dependent populations of the resulting oligomeric species. Higher‐order oligomerization of SPOP stimulates CRL3SPOP ubiquitination efficiency for its physiological substrate Gli3, suggesting that nuclear speckles are hotspots of ubiquitination. Dynamic, higher‐order protein self‐association may be a general mechanism to concentrate functional components in membrane‐less cellular bodies.

Endometrial cancer-associated mutants of SPOP are defective in regulating estrogen receptor-α protein turnover.

Increasing amounts of evidence strongly suggests that dysregulation of ubiquitin-proteasome system is closely associated with cancer pathogenesis. Speckle-type POZ protein (SPOP) is an adapter protein of the CUL3-based E3 ubiquitin ligase complexes. It selectively recruits substrates for their ubiquitination and subsequent degradation. Recently, several exome-sequencing studies of endometrial cancer revealed high frequency somatic mutations in SPOP (5.7–10%). However, how SPOP mutations contribute to endometrial cancer remains unknown. Here, we identified estrogen receptor-α (ERα), a major endometrial cancer promoter, as a substrate for the SPOP-CUL3-RBX1 E3 ubiquitin ligase complex. SPOP specifically recognizes multiple Ser/Thr (S/T)-rich degrons located in the AF2 domain of ERα, and triggers ERα degradation via the ubiquitin-proteasome pathway. SPOP depletion by siRNAs promotes endometrial cells growth. Strikingly, endometrial cancer-associated mutants of SPOP are defective in regulating ERα degradation and ubiquitination. Furthermore, we found that SPOP participates in estrogen-induced ERα degradation and transactivation. Our study revealed novel molecular mechanisms underlying the regulation of ERα protein homeostasis in physiological and pathological conditions, and provided insights in understanding the relationship between SPOP mutations and the development of endometrial cancer.

Speckle-type POZ protein mutations in prostate cancer: a new part of the mosaic.

Speckle-type POZ protein mutations in prostate cancer: a new part of the mosaic.

Abstract 2244: SPOP mutations in prostate cancer across demographically diverse patient cohorts

Abstract Background Recurrent mutations in the Speckle-Type POZ Protein (SPOP) gene have been reported in up to 15% of prostate cancers. However, the frequency and features of cancers with these mutations across different populations is unclear. In addition, detection of point mutations in archival samples can be technically challenging, making mutational analysis of well annotated archival tissue difficult. We set out to investigate SPOP mutations across diverse cohorts, and validate a series of assays employing High Resolution Melting Analysis (HRM) and Sanger sequencing for mutational analysis of FFPE material. Methods: An assay employing HRM and Sanger sequencing was optimized to screen for somatic mutations in recurrently altered areas of the SPOP gene. 720 prostate cancer samples from six international cohorts spanning Caucasian, African American, and Asian patients, including both PSA-screened and unscreened populations, were screened for their SPOP mutation status. Status of SPOP was correlated to molecular features (ERG rearrangement, PTEN deletion, CHD1 deletion) as well as clinical and pathologic features. Results: The overall frequency of SPOP mutations was 8.1% in 720 patient's samples, ranging from 4.6% in the African American cohort to 14.4% in the WCMC cohort. There were no significant differences in frequency between ethnicities or cohorts (p=0.14). SPOP mutation was inversely associated with ERG rearrangement (p<0.01), and SPOP mutant cancers had higher rates of CHD1 deletions (p<0.01). There were no significant differences in rates or time to biochemical recurrence (BCR) in SPOP wild-type (wt) vs mutants (p=0.18, 0.30 respectively). The mutational assays showed excellent sensitivity and specificity in high quality samples. Limitations of this study include missing mutational data due to sample quality and lack of power to identify a difference in clinical outcomes. Conclusions: In this study we were able to show the high sensitivity of HRM followed by Sanger Sequencing to detect mutated SPOP in 720 cases consisting of fresh frozen as well as FFPE material. Using next generation sequencing did not rescue samples which were determined as “assay failure” by HRM and or Sanger Sequencing. SPOP is mutated in 4.6-14.4% of prostate cancer patients across different ethnic and demographic backgrounds. There was no significant association between SPOP mutations with ethnicity, clinical or pathologic parameters. Mutual exclusivity of SPOP mutation with ERG rearrangement as well as a high association with CHD1 deletion reinforces SPOP mutation as defining a distinct molecular subclass of prostate cancer. Citation Format: Mirjam Blattner, Daniel Lee, Catherine O´Reilly, Kyung Park, Theresa Y. MacDonald, Francesca Khani, Kevin Turner, Peter J. Wild, Haley Hieronymus, Charles L. Sawyers, Ashutosh K. Tewari, Holger Moch, Ghil Suk Yoon, Ove Andrén, Katja Fall, Juan Miguel Mosquera, Brian D. Robinson, Andrea Sboner, Christopher E. Barbierie, Mark A. Rubin. SPOP mutations in prostate cancer across demographically diverse patient cohorts. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2244. doi:10.1158/1538-7445.AM2014-2244

Destruction of DDIT3/CHOP protein by wild-type SPOP but not prostate cancer-associated mutants.

Characterization of the exome and genome of prostate cancers by next-generation sequencing has identified numerous genetic alternations. SPOP (speckle-type POZ protein) was identified as one of the most frequently affected genes by somatic point mutations in prostate cancer, suggesting SPOP is potentially a key driver for prostate cancer development and progression. However, how SPOP mutations contribute to prostate cancer remains to be elucidated. SPOP acts as an adaptor protein of the CUL3-RBX1 E3 ubiquitin ligase complex and selectively recruits substrates for their ubiquitination and subsequent degradation. DDIT3 is an endoplasmic reticulum (ER) stress-responsive transcription factor playing an essential role in apoptotic execution pathways triggered by ER stress. Here, we identified DDIT3/CHOP as a bona fide substrate for the SPOP-CUL3-RBX1 E3 ubiquitin ligase complex. SPOP recognizes a Ser/Thr-rich degron in the transactivation domain of DDIT3 and triggers DDIT3 degradation via the ubiquitin-proteasome pathway. Strikingly, prostate cancer-associated mutants of SPOP are defective in promoting DDIT3 degradation. This study reveals novel molecular events underlying the regulation of DDIT3 protein homeostasis and provides insight in understanding the relationship between SPOP mutations and ER stress dysregulation in prostate cancer.