Speckle-type POZ Protein Mutations Research Articles

Co-occurring BRCA2/SPOP Mutations Predict Exceptional Poly (ADP-ribose) Polymerase Inhibitor Sensitivity in Metastatic Castration-Resistant Prostate Cancer

Background and objectiveBRCA2 mutations in metastatic castration-resistant prostate cancer (mCRPC) confer sensitivity to poly (ADP-ribose) polymerase (PARP) inhibitors. However, additional factors predicting PARP inhibitor efficacy in mCRPC are needed. Preclinical studies support a relationship between speckle-type POZ protein (SPOP) inactivation and PARP inhibitor sensitivity. We hypothesized that SPOP mutations may predict enhanced PARP inhibitor response in BRCA2-altered mCRPC. MethodsWe conducted a multicenter retrospective study involving 13 sites. We identified 131 patients with BRCA2-altered mCRPC treated with PARP inhibitors, 14 of which also carried concurrent SPOP mutations. The primary efficacy endpoint was prostate-specific antigen (PSA) response rate (≥50% PSA decline). The secondary endpoints were biochemical progression-free survival (PSA-PFS), clinical/radiographic progression-free survival (PFS), and overall survival (OS). These were compared by multivariable Cox proportional hazard models adjusting for age, tumor stage, baseline PSA level, Gleason sum, prior therapies, BRCA2 alteration types, and co-occurring mutations. Key findings and limitationsBaseline characteristics were similar between groups. PSA responses were observed in 60% (70/117) of patients with BRCA2mut/SPOPwt disease and in 86% (12/14) of patients with BRCA2mut/SPOPmut disease (p = 0.06). The median time on PARP inhibitor treatment was 24.0 mo (95% confidence interval [CI] 19.2 mo to not reached) in this group versus 8.0 mo (95% CI 6.1–10.9 mo) in patients with BRCA2 mutation alone (p = 0.05). In an unadjusted analysis, patients with BRCA2mut/SPOPmut disease experienced longer PSA-PFS (hazard ratio [HR] 0.33 [95% CI 0.15–0.72], p = 0.005) and clinical/radiographic PFS (HR 0.4 [95% CI 0.18–0.86], p = 0.02), and numerically longer OS (HR 0.4 [95% CI 0.15–1.12], p = 0.08). In a multivariable analysis including histology, Gleason sum, prior taxane, prior androgen receptor pathway inhibitor, stage, PSA, BRCA2 alteration characteristics, and other co-mutations, patients with BRCA2mut/SPOPmut disease experienced longer PSA-PFS (HR 0.16 [95% CI 0.05–0.47], adjusted p = 0.001), clinical/radiographic PFS (HR 0.28 [95% CI 0.1–0.81], adjusted p = 0.019), and OS (HR 0.19 [95% CI 0.05–0.69], adjusted p = 0.012). In a separate cohort of patients not treated with a PARP inhibitor, there was no difference in OS between patients with BRCA2mut/SPOPmut versus BRCA2mut/SPOPwt disease (HR 0.97 [95% CI 0.40–2.4], p = 0.94). In a genomic signature analysis, Catalog of Somatic Mutations in Cancer (COSMIC) SBS3 scores predictive of homologous recombination repair (HRR) defects were higher for BRCA2mut/SPOPmut than for BRCA2mut/SPOPwt disease (p = 0.04). This was a retrospective study, and additional prospective validation cohorts are needed. Conclusions and clinical implicationsIn this retrospective analysis, PARP inhibitors appeared more effective in patients with BRCA2mut/SPOPmut than in patients with BRCA2mut/SPOPwt mCRPC. This may be related to an increase in HRR defects in coaltered disease. Patient summaryIn this study, we demonstrate that co-alteration of both BRCA2 and SPOP predicts superior clinical outcomes to treatment with poly (ADP-ribose) polymerase (PARP) inhibitors than BRCA2 alteration without SPOP mutation.

SPOP promotes CREB5 ubiquitination to inhibit MET signaling in liver cancer

Liver cancer is ranked as the sixth most prevalent from of malignancy globally and stands as the third primary contributor to cancer-related mortality. Metastasis is the main reason for liver cancer treatment failure and patient deaths. Speckle-type POZ protein (SPOP) serves as a crucial substrate junction protein within the cullin-RING E3 ligase complex, acting as a significant tumor suppressor in liver cancer. Nevertheless, the precise molecular mechanism underlying the role of SPOP in liver cancer metastasis remain elusive. In the current study, we identified cAMP response element binding 5 (CREB5) as a novel SPOP substrate in liver cancer. SPOP facilitates non-degradative K63-polyubiquitination of CREB5 on K432 site, consequently hindering its capacity to activate receptor tyrosine kinase MET. Moreover, liver cancer-associated SPOP mutant S119N disrupts the SPOP-CREB5 interactions and impairs the ubiquitination of CREB5.This disruption ultimately leads to the activation of the MET signaling pathway and enhances metastatic properties of hepatoma cells both in vitro and in vivo. In conclusion, our findings highlight the functional significance of the SPOP-CREB5-MET axis in liver cancer metastasis.

Opposing Roles of SPOP Mutations in Human Prostate and Endometrial Cancers.

Recurrent gene mutations in speckle-type POZ protein (SPOP), the substrate-binding component of E3 ubiquitin ligase, are associated with tumor progression in prostate and endometrial cancers. Here, we characterized SPOP mutations in these cancers and explored their association with molecular and immune signatures and patient outcomes. There were 7,398 prostate cancer and 19,188 endometrial cancer samples analyzed for clinical and molecular profiles at Caris Life Sciences. Overall survival (OS) was analyzed using Kaplan-Meier survival curves. Statistical significance was determined using chi-square and Mann-Whitney U tests, with P values adjusted for multiple comparisons. SPOP mutations were identified in 9.2% of prostate and 4.3% of endometrial cancers. Mutations clustered in the SPOP meprin and TRAF-C homology domain, with no significant overlap between cancer types. SPOP mutation was associated with differential comutation profiles and opposing tumor immune microenvironment signatures for each cancer, with greater immune infiltration in SPOP-mutated endometrial cancer. SPOP-mutated prostate and endometrial cancers displayed altered epigenetic gene expression, including opposite regulation of BRD2 transcripts. In SPOP-mutant prostate cancer, higher expression of androgen receptor-regulated transcripts and improved OS after treatment with hormonal agents were observed. In endometrial cancer, hormone receptor expression was significantly lower in SPOP-mutated tumors and differences in OS were highly dependent on the particular hotspot mutation and histologic subtype. These data indicate that SPOP mutations drive opposing molecular and immune landscapes in prostate and endometrial cancers-suggesting a loss-of-function mechanism in prostate cancer and gain-of-function mechanism in endometrial cancer-and provide a rationale for tailored therapeutic approaches.

SPOP Mutations Target STING1 Signaling in Prostate Cancer and Create Therapeutic Vulnerabilities to PARP Inhibitor-Induced Growth Suppression.

Speckle-type POZ protein (SPOP) is important in DNA damage response (DDR) and maintenance of genomic stability. Somatic heterozygous missense mutations in the SPOP substrate-binding cleft are found in up to 15% of prostate cancers. While mutations in SPOP predict for benefit from androgen receptor signaling inhibition (ARSi) therapy, outcomes for patients with SPOP-mutant (SPOPmut) prostate cancer are heterogeneous and targeted treatments for SPOPmut castrate-resistant prostate cancer (CRPC) are lacking. Using in silico genomic and transcriptomic tumor data, proteomics analysis, and genetically modified cell line models, we demonstrate mechanistic links between SPOP mutations, STING signaling alterations, and PARP inhibitor vulnerabilities. We demonstrate that SPOP mutations are associated with upregulation of a 29-gene noncanonical (NC) STING (NC-STING) signature in a subset of SPOPmut, treatment-refractory CRPC patients. We show in preclinical CRPC models that SPOP targets and destabilizes STING1 protein, and prostate cancer-associated SPOP mutations result in upregulated NC-STING-NF-κB signaling and macrophage- and tumor microenvironment (TME)-facilitated reprogramming, leading to tumor cell growth. Importantly, we provide in vitro and in vivo mechanism-based evidence that PARP inhibitor (PARPi) treatment results in a shift from immunosuppressive NC-STING-NF-κB signaling to antitumor, canonical cGAS-STING-IFNβ signaling in SPOPmut CRPC and results in enhanced tumor growth inhibition. We provide evidence that SPOP is critical in regulating immunosuppressive versus antitumor activity downstream of DNA damage-induced STING1 activation in prostate cancer. PARPi treatment of SPOPmut CRPC alters this NC-STING signaling toward canonical, antitumor cGAS-STING-IFNβ signaling, highlighting a novel biomarker-informed treatment strategy for prostate cancer.

Abstract A008: Elucidating the effects of the Alzheimer's disease associated gene BIN1 on cancer tumorigenesis

Abstract Purpose- BIN1 is in deleted in ~5% of all prostate cancer (PC) patients and interestingly is highly enriched in the Speckle-type POZ protein (SPOP) mutant subclass (15%) of PC. BIN1 was originally identified as a repressor of cMYC activity and our lab has shown that patients with SPOPmut;MYChigh PC have worse clinical outcomes. Our observation of BIN1 deletion in the SPOP mutant subclass leads us to hypothesize that BIN1 deletion leads to enhanced AR signaling and more aggressive PC. Experimental Procedures- We analyzed baseline protein and mRNA levels of BIN1 in 13 PC cell lines via quantitative polymerase chain reaction (qPCR) and immunoblot. To understand the role of BIN1 in PC cells, we overexpressed BIN1 and evaluated changes in cell proliferation, migration, and AR target gene expression. To elucidate the role of BIN1 in vivo prostate development, we also generated a prostate-specific knockout of BIN1 and investigated changes in prostate development and AR signaling axis over time in this mouse model. Results- Our results show that none of the 13 PC cell lines analyzed had strong protein expression at baseline, but all had detectable mRNA levels. Interestingly, the three cell lines with the highest mRNA expression were AR negative (DU-145), AR independent (LN95) or castration resistant (MDVR). We also saw a decrease in downstream AR target genes (KLK3 and NKX3.1) measured using qPCR after BIN1 overexpression in 22Rv1 cells. We further corroborated this finding by measuring KLK3 expression using a KLK3-luc reporter assay after BIN1 overexpression. BIN1 overexpression also reduced invasive activity of 22Rv1 measured using a Matrigel invasion assay system. Since BIN1 deletion was enriched in SPOP mutant PC clinical data, we evaluated changes in cell proliferation in the wildtype and mutant SPOP cell lines with and without BIN1OE and found that BIN1OEhindered growth of SPOP mutant PC cells. Analysis of the TCGA primary PC dataset illustrate that PC patients harboring BIN1 deletion had higher AR activity compared to PC patients expressing wildtype BIN1. Importantly, mice with prostate specific BIN1 deletion had larger prostate mass at 2 months of age compared to age matched controls and several mice at the 6&9 month had severely enlarged prostates. Conclusion- Our findings show for the first time that BIN1 functions as an inhibitor of the AR signaling axis and deletion of BIN1 leads to higher levels of AR signaling. We also show that BIN1 deletion in mice affects prostate development and leads to enlarged prostates. These novel findings illustrate the clinical significance of BIN1 deletion in PC patients and highlights the AR-signaling axis as a potential target for PC patients with BIN1 deletion Citation Format: Collin McColl, Darlene Skapura, Elisa Echartea, Jenny Deng, Cristian Coarfa, Salma Kaochar, Brian Simons, Aleksandra Rusin. Elucidating the effects of the Alzheimer's disease associated gene BIN1 on cancer tumorigenesis [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A008.

Abstract B069: SPOP loss places the prostate luminal epithelial cells at a selective disadvantage

Abstract Background: Prostate cancer (PC) is the single most common and second-most lethal cancer in men, with over 268,000 estimated cases and over 34,500 estimated deaths in the US in 2022. The Speckle-Type POZ protein (SPOP) mutant subclass of PC accounts for 10% to 15% of all primary PC cases. SPOP is an adaptor for Cullin3/Ring (CUL3-RING)-type E3 ubiquitin ligase complexes and provides substrate specificity. The Cancer Genome Atlas (TCGA) studies show that SPOP is the most frequently mutated gene in primary prostate cancer (PC). Interestingly, PC-associated SPOP mutations are always missense and occur in a heterozygous fashion. The current gap in knowledge is the lack of understanding of the role of wildtype SPOP in PC. Methods: By utilizing prostate specific SPOP knockout (KO) mice, we recently reported increased levels of AR and MYC protein and increased cellular turnover (both proliferation and apoptosis) in the prostate luminal epithelium compared to wildtype prostates. We now characterized these mice for the expression of Cre protein and SPOP mRNA at different age using immunohistochemistry and RNA in situ hybridization. Furthermore, we performed RNA-sequencing analysis in the SPOP knockout mice and matched control littermates. Moreover, we performed RNA-seq in LNCaP, LNCaP-Abl, and RWPE1 cells following SPOP inhibition via siRNA targeting SPOP. Finally, we compared our SPOP inhibition signature from in vitro cell lines and prostate specific SPOP knockout murine model to gain insights about the role of wildtype SPOP protein in the prostate epithelium. Result: Using our Spopfl/fl;PBCre+ model, we observed SPOP floxed cells are rapidly lost and the murine prostate epithelium was repopulated with SPOP wildtype carrying cells. Similarly, knockdown (KD) of SPOP through siRNA treatment in a panel of PC cell lines resulted in a significant reduction in cell viability. These observations suggest that SPOP is important for the normal prostate cell viability. Further transcriptomic profiling of SPOP KO (from transgenic murine model) as well as siSPOP treated in vitro prostate cell lines revealed a significant reduction in the transcriptional activity of the AR. Conclusion: Our data illustrate for the first time a critical role for SPOP in the growth and survival of the prostate epithelium and prostate cancer cell. Our findings further validates SPOP as a important therapeutic target for the treatment of prostate cancer. Citation Format: Kinza Rizwan, Darlene Skapura, Cammy Mason, Cristian Coarfa, Nicholas Mitsiades, Damian Young, Salma Kaochar. SPOP loss places the prostate luminal epithelial cells at a selective disadvantage [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B069.

Abstract 1705: SPOP: An essential gene for normal and prostate tumor cells

Abstract Background: Prostate cancer (PC) is the single most common and second-most lethal cancer in men, with over 268,000 estimated cases and over 34,500 estimated deaths in the US in 2022. The Speckle-Type POZ protein (SPOP) mutant subclass of PC accounts for 10% to 15% of all primary PC cases. SPOP is an adaptor for Cullin3/Ring (CUL3-RING)-type E3 ubiquitin ligase complexes and provides substrate specificity. The Cancer Genome Atlas (TCGA) studies show that SPOP is the most frequently mutated gene in primary prostate cancer (PC). Interestingly, PC-associated SPOP mutations are always missense and occur in a heterozygous fashion. The current gap in knowledge is the lack of understanding of the role of wildtype SPOP in PC. Methods: By utilizing prostate specific SPOP knockout (KO) mice, we recently reported increased levels of AR and MYC protein and increased cellular turnover (both proliferation and apoptosis) in the prostate luminal epithelium compared to wildtype prostates. We now characterized these mice for the expression of Cre protein and SPOP mRNA at different age using immunohistochemistry and RNA in situ hybridization. Furthermore, we performed RNA-sequencing analysis in the SPOP knockout mice and matched control littermates. Moreover, we performed RNA-seq in LNCaP, LNCaP-Abl, and RWPE1 cells following SPOP inhibition via siRNA targeting SPOP. Finally, we compared our SPOP inhibition signature from in vitro cell lines and prostate specific SPOP knockout murine model to gain insights about the role of wildtype SPOP protein in the prostate epithelium. Result: Using our Spopfl/fl;PBCre+ model, we observed SPOP floxed cells are rapidly lost and the murine prostate epithelium was repopulated with SPOP wildtype carrying cells. Similarly, knockdown (KD) of SPOP through siRNA treatment in a panel of PC cell lines resulted in a significant reduction in cell viability. These observations suggest that SPOP is important for the normal prostate cell viability. Further transcriptomic profiling of SPOP KO (from transgenic murine model) as well as siSPOP treated in vitro prostate cell lines revealed a significant reduction in the transcriptional activity of the AR. Conclusion: Our data illustrate for the first time a critical role for SPOP in the growth and survival of the prostate epithelium and prostate cancer cell. Our findings further validates SPOP as a important therapeutic target for the treatment of prostate cancer. Citation Format: Kinza Rizwan, Darlene Skapura, Cammy Mason, Cristian Coarfa, Nicholas Mitsiades, Damian Young, Salma Kaochar. SPOP: An essential gene for normal and prostate tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1705.

Role of adenomatous polyposis coli (APC) gene in SPOP-mutant prostate cancer and clinical outcomes.

202 Background: Speckle-type POZ protein (SPOP) mutations are a frequent initiating event in prostate cancer (PC) and are associated with increased androgen receptor transcriptional output. While SPOP-Mutant (MT) PCs may be more sensitive to hormonal therapy, they are still common amongst castration-resistant PCs (CRPCs) and frequently lethal. It remains unknown which SPOPMT PCs will respond well to hormonal therapy, and which have the potential to develop early castration resistance and cause patient death. Methods: With IRB approval, we retrospectively analyzed NGS data obtained via Tempus|xT tissue assay (DNA sequencing of 648 genes in tumor and matched normal samples at 500x depth) and/or Tempus|xF liquid biopsy assay (ctDNA sequencing of 105 genes in peripheral blood samples at 5,000x depth) for germline and/or somatic mutations in 234 consecutive patients with high-risk localized, locally advanced or metastatic PC followed at Ben Taub Hospital (BTH), a public hospital serving a racially/ethnically diverse patient population (>50% of PC patients are African American). For confirmation, we examined de-identified NGS data from a nationwide cohort of 8812 PC patients sequenced with xT and/or xF by Tempus Labs (Chicago, IL), as well as publicly available data from four additional PC cohorts (PMIDs: 26544944, 34667026, 31061129 and 26855148). Results: We found SPOP mutations in 31/234 BTH PC patients (13.2%). Eleven of these 31 patients (35.5%) also exhibited loss-of-function (LOF) mutations or deletions in the tumor suppressor gene Adenomatous polyposis coli (APC). APC LOF events were found only in 15/203 (7.4%) of the SPOP-wild type (WT) PCs, and the enrichment of SPOPMT PCs for APC LOF events was highly significant (Chi square with Yates correction: P<0.00001). The co-mutation pattern between these two genes was observed even when the analysis was limited specifically to the tissue NGS (133 patients) or the liquid biopsy NGS subsets of the dataset, respectively. This enrichment was confirmed in the nationwide TEMPUS cohort and in the four publicly available clinical PC datasets (APC LOF found in 23.4, 27.0, 21.5, 32.0 and 30.8% of SPOPMT PCs, respectively; and only in 5.8, 2.4, 6.0, 6.7 and 1.0% of SPOP-Wild Type (WT) PCs, respectively (P<0.001 in each cohort). In the BTH PC cohort, SPOPMT PCs harboring APC LOF mutations were more likely to present with metastatic disease (M1) at diagnosis and to develop castration-resistance within 1 year of first-line hormonal therapy than SPOPMT PCs with WT APC. Conclusions: In SPOPMT PCs, inactivation of APC is a common event and associated with more aggressive disease (higher metastatic potential and faster progression to castration resistance). This observation may help identify SPOPMT PC patients who are at higher risk for early castration resistance and could benefit from more intensive treatment.

Higher-order SPOP assembly reveals a basis for cancer mutant dysregulation

The speckle-type POZ protein (SPOP) functions in the Cullin3-RING ubiquitin ligase (CRL3) as a receptor for the recognition of substrates involved in cell growth, survival, and signaling. SPOP mutations have been attributed to the development of many types of cancers, including prostate and endometrial cancers. Prostate cancer mutations localize in the substrate-binding site of the substrate recognition (MATH) domain and reduce or prevent binding. However, most endometrial cancer mutations are dispersed in seemingly inconspicuous solvent-exposed regions of SPOP, offering no clear basis for their cancer-causing and peculiar gain-of-function properties. Herein, we present the first structure of SPOP in its oligomeric form, uncovering several new interfaces important for SPOP self-assembly and normal function. Given that many previously unaccounted-for cancer mutations are localized in these newly identified interfaces, we uncover molecular mechanisms underlying dysregulation of SPOP function, with effects ranging from gross structural changes to enhanced self-association, and heightened stability and activity.

Deregulation of SPOP in Cancer.

Speckle-type POZ protein (SPOP) is a substrate-binding adaptor of the CULLIN3/RING-box1 E3 ubiquitin ligase complex. SPOP is frequently mutated in prostate and endometrial cancers, whereas it is overexpressed in renal cell carcinoma (RCC). SPOP can mediate both degradable and nondegradable polyubiquitination of a number of substrates with diverse biological functions such as androgen receptor (AR), SRC-3, TRIM24, BRD4, PD-L1, 53BP1, GLP/G9a, c-Myc, SENP7, among others. Cancer-associated SPOP mutants often impair SPOP binding and polyubiquitination of its substrates to influence various cancer-relevant pathways, which include androgen/AR signaling, DNA repair and methylation, cellular stress surveillance, cancer metabolism, and immunity. Although SPOP is recognized as a tumor suppressor in prostate and endometrial cancers, it acts like an oncoprotein in RCC. This review provides an overview of the recent progress in understanding of the upstream regulators of SPOP and its downstream targets, highlights the significant impact of SPOP mutations and overexpression on cancer pathogenesis, and discusses the potential of targeting SPOP for cancer treatment.

Selected Articles from This Issue

Malignant pleural mesothelioma (MPM) is a highly aggressive cancer that lacks effective therapeutic options, owing to chemotherapeutic resistance and lack of currently targetable oncogenic mutations. However, mutations in histone modifiers documented in Mp. represent exciting potential therapeutic targets. Wirawan and colleagues used estrogen receptor sensitive Snail variant mesenchymal and sarcomatoid-like MPM cell lines to show that lysine-specific demethylase 1 (LSD1) drives a MPM mesenchymal phenotype, tumor cell migration, and cisplatin resistance. The authors integrated microarray, phospho-kinase array, and assay for transposase-accessible chromatin using sequencing (ATAC-seq) analyses to identify milk fat globulin protein E8 (MFGE8) as the LSD1-mediated driver of cisplatin resistance. Subsequent experiments demonstrated that MFGE8 interaction with integrin β3 drives FAK and AKT phosphorylation, initiating a positive feedback loop that upregulates Snail expression and promotes its associated mesenchymal phenotype. In sum, the work presented suggests that LSD1 may be a multifactorial therapeutic target for MPM.While androgen deprivation can provide at least temporary benefit for men with metastatic prostate cancer, most patients go on to develop lethal castration-resistant prostate cancer (CRPC). Speckle-type POZ protein (SPOP) mutations and Sonic hedgehog (SHH)-mediated androgen receptor (AR) signaling activation are prevalent in CRPC, making each therapeutic targets of interest for the disease. In their study, Burleson and colleagues demonstrate that a transcriptional activator of the SHH pathway, GLI3, enhances SHH signaling and cell proliferation in androgen-deprived prostate cancer cells. The authors show that GLI3 is post-translationally stabilized by somatic SPOP mutations, and that stabilized GLI3 partners with AR to induce a gene expression program that promotes tumor cell proliferation in vitro and in vivo. CRPC patients whose tumors expressed the GLI3 gene signature displayed higher biochemical relapse rates, suggesting that the signature may identify patients at high risk of disease progression. Overall, the presented data provide a novel mechanistic link between SPOP mutations and SHH-mediated AR signaling activation that could inform prognostic and therapeutic strategies for CRPC moving forward.The efficacy of antiangiogenic cancer therapies has been limited by side effects and therapeutic resistance, suggesting that targeting angiogenesis alongside other oncogenic processes may be more effective than targeting angiogenesis alone. Recent work linking low IQ motif-containing GTPase-activating protein 2 (IQGAP2) expression to breast tumor epithelial–mesenchymal transition (EMT), angiogenesis, and poor patient outcomes makes IQGAp. an intriguing therapeutic candidate. Kumar and colleagues used IQGAP2 ablation and overexpression in both estrogen receptor-positive and -negative breast cancer cell lines to demonstrate that IQGAP2 reduction in those cell lines increases endothelial cell proliferation and migration. Accordingly, IQGAP2 reduction increased vascular density in chick chorioallantoic membranes and wound healing assays, which supported the authors' observation that IQGAP2 expression inversely correlated with endothelial cell abundance in tumor cells from breast cancer patients. The authors mechanistically attributed their findings to increased ERK-VEGF-A signaling in tumor cells, which activated VEGFR2-AKT signaling in endothelial cells. Results from the study position IQGAP2 as a next-generation anti-angiogenic therapeutic target potentially capable of simultaneously inhibiting angiogenesis and EMT.Draining lymph node metastasis is often noted upon nasopharyngeal carcinoma (NPC) diagnosis, and can mark an initial stage of progressive metastatic dissemination. While Epstein-Barr virus (EBV) has long been associated with NPC, its role in initial metastatic processes has not been defined. In their study, Li and colleagues used EBV-infected and functional EBV knockout NPC cell lines to demonstrate that EBV-positive NPC induces lymphatic endothelial cell proliferation and migration. Gene expression analysis revealed elevated VEGF-C expression in NPC infected with EBV, and the authors showed that VEGF-C was responsible for EBV-induced lymphangiogenesis. Further work demonstrated that EBV miRNA BART15 targets PHLPP1 in NPC cells, resulting in increased AKT phosphorylation, HIF-1α activation, and VEGF-C production. Moreover, patients with EBV-positive NPC displayed higher levels of lymphangiogenesis and lymph node metastasis. Taken together, the presented work sheds light on how EBV induces early NPC progression and elucidates a molecular pathway that could be therapeutically targeted.

GLI3 Is Stabilized by SPOP Mutations and Promotes Castration Resistance via Functional Cooperation with Androgen Receptor in Prostate Cancer.

Although the Sonic hedgehog (SHH) signaling pathway has been implicated in promoting malignant phenotypes of prostate cancer, details on how it is activated and exerts its oncogenic role during prostate cancer development and progression is less clear. Here, we show that GLI3, a key SHH pathway effector, is transcriptionally upregulated during androgen deprivation and posttranslationally stabilized in prostate cancer cells by mutation of speckle-type POZ protein (SPOP). GLI3 is a substrate of SPOP-mediated proteasomal degradation in prostate cancer cells and prostate cancer driver mutations in SPOP abrogate GLI3 degradation. Functionally, GLI3 is necessary and sufficient for the growth and migration of androgen receptor (AR)-positive prostate cancer cells, particularly under androgen-depleted conditions. Importantly, we demonstrate that GLI3 physically interacts and functionally cooperates with AR to enrich an AR-dependent gene expression program leading to castration-resistant growth of xenografted prostate tumors. Finally, we identify an AR/GLI3 coregulated gene signature that is highly correlated with castration-resistant metastatic prostate cancer and predictive of disease recurrence. Together, these findings reveal that hyperactivated GLI3 promotes castration-resistant growth of prostate cancer and provide a rationale for therapeutic targeting of GLI3 in patients with castration-resistant prostate cancer (CRPC). IMPLICATIONS: We describe two clinically relevant mechanisms leading to hyperactivated GLI3 signaling and enhanced AR/GLI3 cross-talk, suggesting that GLI3-specific inhibitors might prove effective to block prostate cancer development or delay CRPC.

SPOP promotes CDCA5 degradation to regulate prostate cancer progression via the AKT pathway.

The E3 ubiquitin ligase adaptor Speckle-type POZ protein (SPOP) plays an important tumour suppressor role in prostate cancers (PCa), with mutation rate up to 15%. However, how SPOP mutations regulate prostate tumorigenesis remains elusive. Here, we report the identification of cell division cycle associated 5 (CDCA5) as a SPOP substrate. We found that SPOP interacts with CDCA5 and promotes its polyubiquitin degradation in a degron-dependent manner. This effect was greatly impaired by introducing PCa associated SPOP mutations. Importantly, we found that CDCA5 was essential for PCa cells to survive and proliferate. CDCA5 depletion in PCa cells led to cessation of proliferation, G2M arrest, severe sister chromatid aggregation disturbance, and apoptosis. we also found that CDCA5 knockdown decreased the protein expression of p-GSK3β, increased the activity of caspase-3, caspase-9, and the Bax/Bcl-2 ratio. Besides, we confirmed that CDCA5 interrupted cancer cell behavior via the AKT pathway. In contrast, silencing SPOP or overexpressing CDCA5 increased cell proliferation. Consistently, depleting SPOP along with CDCA5, or overexpressing CDCA5 along with SPOP also caused the growth of cells repressed. Consistent with the functional role of CDCA5, the mRNA and protein levels of CDCA5 were significantly increased in PCa, compared to normal tissues, and its high expression was associated with more severe lymph node metastasis, higher Gleason score, and poorer prognosis. Together, our data showed that SPOP plays a crucial role in inhibiting tumorigenesis and partly achieved this by promoting the degradation of oncoprotein CDCA5.

SPOP-mediated ubiquitination and degradation of PDK1 suppresses AKT kinase activity and oncogenic functions

Background3-phosphoinositide-dependent protein kinase-1 (PDK1) acts as a master kinase of protein kinase A, G, and C family (AGC) kinase to predominantly govern cell survival, proliferation, and metabolic homeostasis. Although the regulations to PDK1 downstream substrates such as protein kinase B (AKT) and ribosomal protein S6 kinase beta (S6K) have been well established, the upstream regulators of PDK1, especially its degrader, has not been defined yet.MethodA clustered regularly interspaced short palindromic repeats (CRISPR)-based E3 ligase screening approach was employed to identify the E3 ubiquitin ligase for degrading PDK1. Western blotting, immunoprecipitation assays and immunofluorescence (IF) staining were performed to detect the interaction or location of PDK1 with speckle-type POZ protein (SPOP). Immunohistochemistry (IHC) staining was used to study the expression of PDK1 and SPOP in prostate cancer tissues. In vivo and in vitro ubiquitination assays were performed to measure the ubiquitination conjugation of PDK1 by SPOP. In vitro kinase assays and mass spectrometry approach were carried out to identify casein kinase 1 (CK1) and glycogen synthase kinase 3 (GSK3)-mediated PDK1 phosphorylation. The biological effects of PDK1 mutations and correlation with SPOP mutations were performed with colony formation, soft agar assays and in vivo xenograft mouse models.ResultsWe identified that PDK1 underwent SPOP-mediated ubiquitination and subsequent proteasome-dependent degradation. Specifically, SPOP directly bound PDK1 by the consensus degron in a CK1/GSK3β-mediated phosphorylation dependent manner. Pathologically, prostate cancer patients associated mutations of SPOP impaired PDK1 degradation and thus activated the AKT kinase, resulting in tumor malignancies. Meanwhile, mutations that occurred around or within the PDK1 degron, by either blocking SPOP to bind the degron or inhibiting CK1 or GSK3β-mediated PDK1 phosphorylation, could markedly evade SPOP-mediated PDK1 degradation, and played potently oncogenic roles via activating the AKT kinase.ConclusionsOur results not only reveal a physiological regulation of PDK1 by E3 ligase SPOP, but also highlight the oncogenic roles of loss-of-function mutations of SPOP or gain-of-function mutations of PDK1 in tumorigenesis through activating the AKT kinase.

The SPOP-ITCH Signaling Axis Protects Against Prostate Cancer Metastasis

Prostate cancer is one of the most common causes of cancer incidence and death in men, with the mortality caused primarily by the late-stage and metastatic forms of the disease. The mechanisms and molecular markers for prostate cancer metastasis are not fully understood. Speckle type Poz Protein (SPOP) is an E3 ubiquitin ligase adaptor that is often mutated in prostate cancer. In this study, we sequenced the SPOP gene in 198 prostate cancer patients and found 16 mutations in the cohort. Multivariate analysis revealed that SPOP mutations correlated with the clinical stage of the disease and strongly with metastasis. We identified ITCH as a candidate protein for SPOP-mediated degradation via mass spectrometry. We demonstrated the interaction between SPOP and ITCH, and found that the SPOP F133L mutation disrupted the SPOP-ITCH interaction, leading to a subsequent increase in the ITCH protein level. Further, we found that the SPOP knockdown led to higher levels of Epithelial- mesenchymal transition (EMT) proteins and increased cell invasion. Together, our results highlight the functional significance of the SPOP-ITCH pathway in prostate cancer metastasis.

SPOP Promotes GLI3‐dependent SHH Signaling in Breast Cancer

Personalized treatment, which is designed to only block oncogenic pathways activated by specific cancer mutations, is rapidly rising as a superior treatment for cancer. One gene that has recently been identified as highly mutated in a variety of cancers is Speckle Type POZ protein (SPOP), a substrate binding subunit of an E3 ubiquitin ligase that has been shown to target Sonic Hedgehog (SHH) downstream effectors for degradation. Interestingly, recent studies have found that SPOP is downregulated in up to 70% of breast cancer tumors and, furthermore, a correlation has been found between high levels of SHH signaling and poor prognostic pathological breast cancer features. Previous studies from our laboratory have shown that SPOP binds directly to the SHH downstream effector GLI3 to target it for degradation in a manner that is disrupted by prostate cancer-associated SPOP mutations. Since SHH signaling has been shown to be a key player in breast cancer oncogenesis, it is likely that SPOP plays a similar role in breast cancer progression. We therefore hypothesize that downregulation of SPOP induces hyper-activated SHH signaling to promote breast cancer progression through stabilization of GLI3. In order to study this, we employed a lentivirus-mediated approach to generate SPOP knockdown breast cancer cells. Next, we carried out western blot analysis to examine GLI3 protein levels when SPOP is downregulated followed by co-immunoprecipitation analysis to examine a GLI3/SPOP interaction. Finally, we used MG132 treatment to block the proteasome pathway in order to determine whether SPOP-mediated degradation of GLI3 functions through the proteasome. Our results indicate that SPOP knockdown promotes upregulation of the SHH downstream effector GLI3 through a direct physical interaction. Furthermore, we show that SPOP likely targets GLI3 for degradation through the proteasome pathway as MG132 treatment promotes GLI3 stabilization even in the presence of SPOP. Our collective results therefore indicate that SPOP targets GLI3 for degradation through the proteasome pathway which, in turn, would cause hyper-activated SHH signaling and subsequent progression of breast cancer oncogenesis.

SPOP mutation as a predictive marker for treatment of metastatic castration-resistant prostate cancer.

160 Background: Predictive markers linking molecular background to treatment response and clinical outcomes are currently lacking in prostate cancer. Missense mutations in SPOP (speckle-type POZ protein) define a distinct molecular subtype, occurring in about 12% of both clinically localized and metastatic disease. SPOP mutations occur early in prostate tumorigenesis and facilitate dysregulation of the androgen-receptor (AR) signaling network, suggesting mutant SPOP can impact response to AR-directed therapies. We hypothesized that SPOP mutation will be associated with superior response to next-generation AR inhibitors in metastatic castration-resistant prostate cancer (mCRPC). Methods: This is a retrospective study to evaluate patients who progressed to mCRPC and were treated with AR-targeted therapy (i.e. enzalutamide, abiraterone acetate). Eligibility criteria inlcuded androgen deprivation therapy, metastatic disease documented by bone lesion or soft tissue identified on imaging, castrate testosterone level ( < 50g/dL), and evaluable tissue for DNA analysis. SPOP status was determined by next-generation sequencing. Time to PSA progression (PSA TTP) was defined per PCWG2 as PSA increase of 25% from nadir and a minimum of 2 ng/ml calculated from date of primary treatment initiation to the date of PSA progression. Overall survival (OS) was calculated from start of treatment to date of death. Statisitcal comparision of the SPOP mutant or wild-type was determined using Kaplan-Meier and independent t-test analysis. Results: The analysis included 69 men with mCRPC with previous or ongoing ADT and receipt of AR-directed therapy (aberaterone acetate, enzalutamide). SPOP status was determined for all patients: 7 patients SPOP mutant, 62 patients SPOP wild-type. Mutant SPOP was associated with significantly longer PSA TTP (22.5 vs. 9.7 months, p = 0.031) and improved OS (21 vs. 29 months, p = 0.12) with enzalutamide or abiraterone as compared to SPOP wild-type patients. Conclusions: Our data demonstrate that SPOP mutations predict for better outcomes with next-generation AR inhibitors in men with mCRPC. SPOP mutations are a prominent molecular sub-type with potential to impact clinical management in men with metastatic, therapy resistant prostate cancer.

CHD1 and SPOP synergistically protect prostate epithelial cells from DNA damage.

Recent genomic profiling has identified a subtype of prostate cancer (PCa) characterized by two key genetic alterations: missense mutation of speckle-type POZ protein (SPOP) and homozygous deletion of chromodomain helicase DNA-binding protein 1 (CHD1). Mutually exclusive with E26 transformation-specific (ETS) rearrangements, this subtype displays high genomic instability. Previous studies indicate that deficient SPOP or CHD1 alone leads to feeble prostate abnormalities and each protein is involved in DNA damage response (DDR). It remains to be determined whether CHD1 and SPOP cooperate to suppress prostate tumorigenesis and DDR. Prostate-specific single or double knockout of Spop and Chd1 was generated with the Cre/loxP system in mice. Wild-type or mutant SPOP (F102C, F133V) overexpression and CHD1 knockdown with short hairpin RNA were created in human benign prostatic hyperplasia cell line BPH1. The levels of DNA damage and homologous recombination repair were measured by immunofluorescence staining of γH2AX and RAD51, respectively. Spop/Chd1 double-knockout mice displayed prostatic intraepithelial neoplasia at both young (3 months) and old (12 months) ages and failed to generate prostate adenocarcinoma. Compared with wild-type or single-knockout mice, the double-knockout prostate harbored moderately higher proliferating cells and dramatically augmented the level of γH2AX staining, although androgen receptor-positive cells and apoptotic cells remained at a similar level. In BPH1 cell line, SPOP mutant overexpression and CHD1 silencing synergistically sensitized the cells to DNA damage by camptothecin, an inducer of double-strand breaks. Our results indicate that SPOP and CHD1 can synergistically promote repair of naturally occurring or chemically induced DNA damages in prostate epithelial cells. Regarding the progression of the SPOP/CHD1 subtype of PCa, other functionally complementary drivers warrant further identification. The clinical implication is that this subtype of PCa may be particularly sensitive to poly(ADP-ribose) polymerase inhibitors or DNA-damaging agents.

Impact of somatic SPOP (Speckle-Type POZ protein) mutation (mtSPOP) on response to systemic therapy and survival outcome in men with de novo metastatic castration-sensitive prostate cancer (d-mCSPC).

329 Background: Men with metastatic castration resistant prostate cancer harboring somatic mutant SPOP (mt SPOP) have improved progression free survival (PFS) on abiraterone than those with wild-type SPOP (wt SPOP) (Boysen et al, CCR 2018; PMID: 30068710). We hypothesized that mtSPOP will be associated with improved response to systemic therapy and outcomes in mCSPC. Methods: This retrospective study included patients from 4 academic institutions. Eligibility criteria: receipt of standard androgen deprivation therapy (ADT) without intensification (chemotherapy or novel hormonal agents) for the diagnosis of d-mCSPC, no prior history or treatment for prostate cancer, and established SPOP status determined by targeted next-generation sequencing. PFS was defined per PCWG2 defined PSA or investigator assessed radiographic progression. Overall survival (OS) was calculated from date of starting ADT for d-mCSPC to date of death. Kaplan-Meier analysis and t-test were used to compare variables in these two cohorts. Results: Of 110 mtSPOP men with advanced prostate cancer identified, 37 had d-mCSPC of which 25 received ADT. Of 353 wtSPOP patients, 184 had d-mCSPC of which 97 received ADT. Baseline demographics and disease characteristic were similar (table). mtSPOP was associated with significantly improved PFS [35 vs. 14 months, HR 0.519 (95% CI 0.312-0.861), p=0.011] and OS [97 vs. 69 months, HR 0.4392 (95% CI=0.207-0.931); p=0.032] with ADT as compared to wtSPOP patients. Conclusions: Men with d-mCSPC with somatic mtSPOP have improved outcomes with ADT than those with wtSPOP. Once validated, these hypothesis generating data may aid with counselling and treatment selection, as well as patient stratification in future trials in d-mCSPC.[Table: see text]

Cancer Mutations of the Tumor Suppressor SPOP Disrupt the Formation of Active, Phase-Separated Compartments

Mutations in the tumor suppressor SPOP (speckle-type POZ protein) cause prostate, breast, and other solid tumors. SPOP is a substrate adaptor of the cullin3-RING ubiquitin ligase and localizes to nuclear speckles. Although cancer-associated mutations in SPOP interfere with substrate recruitment to the ligase, mechanisms underlying assembly of SPOP with its substrates in liquid nuclear bodies and effects of SPOP mutations on assembly are poorly understood. Here, we show that substrates trigger phase separation of SPOP invitro and co-localization in membraneless organelles in cells. Enzymatic activity correlates with cellular co-localization and invitro mesoscale assembly formation. Disease-associated SPOP mutations that lead to the accumulation of proto-oncogenic proteins interfere with phase separation and co-localization in membraneless organelles, suggesting that substrate-directed phase separation of this E3 ligase underlies the regulation of ubiquitin-dependent proteostasis.