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KDM5B (lysine[K]-specific demethylase 5B) is frequently upregulated in various human cancers including prostate cancer. KDM5B controls H3K4me3/2 levels and regulates gene transcription and cell differentiation, yet the contributions of KDM5B to prostate cancer tumorigenesis remain unknown. In this study, we investigated the functional role of KDM5B in epigenetic dysregulation and prostate cancer progression in cultured cells and in mouse models of prostate epithelium–specific mutant Pten/Kdm5b. Kdm5b deficiency resulted in a significant delay in the onset of prostate cancer in Pten-null mice, whereas Kdm5b loss alone caused no morphologic abnormalities in mouse prostates. At 6 months of age, the prostate weight of Pten/Kdm5b mice was reduced by up to 70% compared with that of Pten mice. Pathologic analysis revealed Pten/Kdm5b mice displayed mild morphologic changes with hyperplasia in prostates, whereas age-matched Pten littermates developed high-grade prostatic intraepithelial neoplasia and prostate cancer. Mechanistically, KDM5B governed PI3K/AKT signaling in prostate cancer in vitro and in vivo. KDM5B directly bound the PIK3CA promoter, and KDM5B knockout resulted in a significant reduction of P110α and PIP3 levels and subsequent decrease in proliferation of human prostate cancer cells. Conversely, KDM5B overexpression resulted in increased PI3K/AKT signaling. Loss of Kdm5b abrogated the hyperactivation of AKT signaling by decreasing P110α/P85 levels in Pten/Kdm5b mice. Taken together, our findings reveal that KDM5B acts as a key regulator of PI3K/AKT signaling; they also support the concept that targeting KDM5B is a novel and effective therapeutic strategy against prostate cancer.Significance:This study demonstrates that levels of histone modification enzyme KDM5B determine hyperactivation of PI3K/AKT signaling in prostate cancer and that targeting KDM5B could be a novel strategy against prostate cancer.

Suppl Figs S1-S8

Table S1, S2 and S3

Supplementary Methods, Figures 1-4 from The G Protein–Coupled Receptor 87 Is Necessary for p53-Dependent Cell Survival in Response to Genotoxic Stress

KDM5B (lysine[K]-specific demethylase 5B) is frequently upregulated in various human cancers including prostate cancer. KDM5B controls H3K4me3/2 levels and regulates gene transcription and cell differentiation, yet the contributions of KDM5B to prostate cancer tumorigenesis remain unknown. In this study, we investigated the functional role of KDM5B in epigenetic dysregulation and prostate cancer progression in cultured cells and in mouse models of prostate epithelium–specific mutant Pten/Kdm5b. Kdm5b deficiency resulted in a significant delay in the onset of prostate cancer in Pten-null mice, whereas Kdm5b loss alone caused no morphologic abnormalities in mouse prostates. At 6 months of age, the prostate weight of Pten/Kdm5b mice was reduced by up to 70% compared with that of Pten mice. Pathologic analysis revealed Pten/Kdm5b mice displayed mild morphologic changes with hyperplasia in prostates, whereas age-matched Pten littermates developed high-grade prostatic intraepithelial neoplasia and prostate cancer. Mechanistically, KDM5B governed PI3K/AKT signaling in prostate cancer in vitro and in vivo. KDM5B directly bound the PIK3CA promoter, and KDM5B knockout resulted in a significant reduction of P110α and PIP3 levels and subsequent decrease in proliferation of human prostate cancer cells. Conversely, KDM5B overexpression resulted in increased PI3K/AKT signaling. Loss of Kdm5b abrogated the hyperactivation of AKT signaling by decreasing P110α/P85 levels in Pten/Kdm5b mice. Taken together, our findings reveal that KDM5B acts as a key regulator of PI3K/AKT signaling; they also support the concept that targeting KDM5B is a novel and effective therapeutic strategy against prostate cancer. Significance: This study demonstrates that levels of histone modification enzyme KDM5B determine hyperactivation of PI3K/AKT signaling in prostate cancer and that targeting KDM5B could be a novel strategy against prostate cancer.

EZH2 is crucial for the progression of prostate cancer (PCa) and castration-resistant prostate cancer (CRPC) through upregulation and activation of progenitor genes, as well as androgen receptor (AR)-target genes. However, the mechanisms by which EZH2 is regulated in PCa and CRPC remain elusive. Here we report that EZH2 is post-transcriptionally regulated by SKP2 in vitro in cultured cells and in vivo in mouse models. We observed aberrant upregulation of Skp2, Ezh2 and histone H3 lysine 27 trimethylation (H3K27me3) in both Pten null mouse embryonic fibroblasts (MEFs) and Pten null mouse prostate tissues. Loss of Skp2 resulted in a striking decrease of Ezh2 levels in Pten/Trp53 double-null MEFs and in prostate tumors of Pten/Trp53 double-null mutant mice. SKP2 knockdown decreased EZH2 levels in human PCa cells through upregulation of TRAF6-mediated and lysine(K) 63-linked ubiquitination of EZH2 for degradation. Ectopic expression of TRAF6 promoted the K63-linked ubiquitination of EZH2 to decrease EZH2 and H3K27me3 levels in PCa cells. In contrast, TRAF6 knockdown resulted in a reduced EZH2 ubiquitination with an increase of EZH2 and H3K27me3 levels in PCa cells. Furthermore, the catalytically dead mutant TRAF6 C70A abolished the TRAF6-mediated polyubiquitination of recombinant human EZH2 in vitro. Most importantly, a concurrent elevation of Skp2 and Ezh2 was found in CRPC tumors of Pten/Trp53 mutant mice, and expression levels of SKP2 and EZH2 were positively correlated in human PCa specimens. Taken together, our findings revealed a novel mechanism on EZH2 ubiquitination and an important signaling network of SKP2-TRAF6-EZH2/H3K27me3, and targeting SKP2-EZH2 pathway may be a promising therapeutic strategy for CRPC treatment.

ARF couples with TP53 in a canonical signaling pathway to activate cellular senescence for tumor suppressive function under oncogenic insults. However, the mechanisms on aberrant elevation of ARF in cancers are still poorly understood. We previously showed that ARF (p14ARF in human and p19Arf in mouse) elevation correlates with PTEN loss and stabilizes SLUG to reduce cell adhesion in prostate cancer (PCa). Here we report that ARF is essential for MMP7 expression, E-Cadherin decrease and the anchorage loss to the extracellular matrix (ECM) in PCa in vitro and in vivo. We found that Mmp7 is aberrantly elevated in cytosol and nucleus of malignant prostate tumors of Pten/Trp53 mutant mice. Interestingly, p19Arf deficiency strikingly decreases Mmp7 levels but increases E-Cadherin in Pten/Trp53/p19Arf mice. ARF knockdown markedly reduces MMP7 in human PCa cells. Conversely, tetracycline-inducible expression of ARF increases MMP7 with a decrease of E-Cadherin in PCa cells. Importantly, MMP7 physically binds ARF to show the co-localization in nucleus. Co-expression of MMP7 and ARF promotes cell migration, and MMP7 knockdown decreases wound healing in PCa cells. Furthermore, MMP7 elevation correlates with ARF expression in advanced human PCa. Our findings reveal for the first time that the crosstalk between ARF and MMP7 in nucleus contributes to ECM network in tumor microenvironments in vivo, implicating a novel therapeutic target for advanced PCa treatment.

Nuclear receptor tyrosine kinases (nRTKs) are aberrantly upregulated in many types of cancers, but the regulation of nRTK remains unclear. We previously showed androgen deprivation therapy (ADT) induces nMET in castration resistant prostate cancer (CRPC) specimens. Through gene expression microarray profiles reanalysis, we identified that nMET signaling requires ARF in CRPC in vivo Arf deficiency in Pten/Trp53 conditional knockout mouse model. Accordingly, aberrant MET/nMET elevation correlates with ARF in human prostate cancer (PCa) specimens. Mechanistically, ARF elevates nMET through binding to MET cytoplasmic domain to stabilize MET. Furthermore, carbon nanodots resensitize cancer cells to MET inhibitors through DNA damage response. The inhibition of phosphorylation by carbon nanodots was identified through binding to phosphate group of phosphory-tyrosine via computational calculation and experimental assay. Thus nMET is essential to precision therapy of MET inhibitor. Our findings reveal for the first time that targeting nMET axis by carbon nanodots can be a novel avenue for overcoming drug resistance in cancers especially prostate cancer.

The identification and targeting of biomarkers specific to prostate cancer (PCa) could improve its detection. Given the high expression of translocator protein (TSPO) in PCa, we investigated the use of [18F]VUIIS1008 (a novel TSPO-targeting radioligand) coupled with positron emission tomography (PET) to identify PCa in mice and to characterize their TSPO uptake. Ptenpc−/−, Trp53pc−/− prostate cancer-bearing mice (n = 9, 4–6 months old) were imaged in a 7T MRI scanner for lesion localization. Within 24 h, the mice were imaged using a microPET scanner for 60 min in dynamic mode following a retro-orbital injection of ~ 18 MBq [18F]VUIIS1008. Following imaging, tumors were harvested and stained with a TSPO antibody. Regions of interest (ROIs) were drawn around the tumor and muscle (hind limb) in the PET images. Time-activity curves (TACs) were recorded over the duration of the scan for each ROI. The mean activity concentrations between 40 and 60 min post radiotracer administration between tumor and muscle were compared. Tumor presence was confirmed by visual inspection of the MR images. The uptake of [18F]VUIIS1008 in the tumors was significantly higher (p

Sirtuins are NAD+-dependent deacetylases that facilitate cellular stress response. They include SirT6, which protects genome stability and regulates metabolic homeostasis through gene silencing, and whose loss induces an accelerated aging phenotype directly linked to hyperactivation of the NF-κB pathway. Here we show that SirT6 binds to the H3K9me3-specific histone methyltransferase Suv39h1 and induces monoubiquitination of conserved cysteines in the PRE-SET domain of Suv39h1. Following activation of NF-κB signaling Suv39h1 is released from the IκBα locus, subsequently repressing the NF-κB pathway. We propose that SirT6 attenuates the NF-κB pathway through IκBα upregulation via cysteine monoubiquitination and chromatin eviction of Suv39h1. We suggest a mechanism based on SirT6-mediated enhancement of a negative feedback loop that restricts the NF-κB pathway., Sirtuins are involved in the regulation of responses to diverse types of cellular stress. Here the authors describe the SirT6-dependent cysteine monoubiquitination of the histone methyltransferase Suv39h1 as part of a regulatory circuit for the NF-κB pathway.

The initiation and progression of human prostate cancer are highly associated with aberrant dysregulations of tumor suppressors and proto-oncogenes. Despite that deletions and mutations of tumor suppressors and aberrant elevations of oncogenes at the genetic level are reported to cause cancers, emerging evidence has revealed that cancer progression is largely regulated by posttranslational modifications (PTMs) and epigenetic alterations. PTMs play critical roles in gene regulation, cellular functions, tissue development, diseases, malignant progression and drug resistance. Recent discoveries demonstrate that ubiquitination and SUMOylation are complicated but highly-regulated PTMs, and make essential contributions to diseases and cancers by regulation of key factors and signaling pathways. Ubiquitination and SUMOylation pathways can be differentially modulated under various stimuli or stresses in order to produce the sustained oncogenic potentials. In this review, we discuss some new insights about molecular mechanisms on ubiquitination and SUMOylation, their associations with diseases, oncogenic impact on prostate cancer (PCa) and clinical implications for PCa treatment.

Copy-number alterations (CNA) are among the most common molecular events in human prostate cancer genomes and are associated with worse prognosis. Identification of the oncogenic drivers within these CNAs is challenging due to the broad nature of these genomic gains or losses which can include large numbers of genes within a given region. Here, we profiled the genomes of four genetically engineered mouse prostate cancer models that reflect oncogenic events common in human prostate tumors, with the goal of integrating these data with human prostate cancer datasets to identify shared molecular events. Met was amplified in 67% of prostate tumors from Pten p53 prostate conditional null mice and in approximately 30% of metastatic human prostate cancer specimens, often in association with loss of PTEN and TP53. In murine tumors with Met amplification, Met copy-number gain and expression was present in some cells but not others, revealing intratumoral heterogeneity. Forced MET overexpression in non–MET-amplified prostate tumor cells activated PI3K and MAPK signaling and promoted cell proliferation and tumor growth, whereas MET kinase inhibition selectively impaired the growth of tumors with Met amplification. However, the impact of MET inhibitor therapy was compromised by the persistent growth of non–Met-amplified cells within Met-amplified tumors. These findings establish the importance of MET in prostate cancer progression but reveal potential limitations in the clinical use of MET inhibitors in late-stage prostate cancer. Mol Cancer Ther; 14(1); 278–88. ©2014 AACR.

// Wenfu Lu 1 , Shenji Liu 1 , Bo Li 1 , Yingqiu Xie 1 , Christine Adhiambo 1 , Qing Yang 1 , Billy R. Ballard 2 , Keiichi I. Nakayama 3 , Robert J. Matusik 4 , Zhenbang Chen 1 1 Department of Biochemistry and Cancer Biology, Meharry Medical College, TN 37208, USA 2 Department of Pathology, Anatomy and Cell Biology, Meharry Medical College, TN 37208, USA 3 Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan 4 Department of Urologic Surgery, Vanderbilt University School of Medicine, TN 37232, USA Correspondence to: Zhenbang Chen, e-mail: zchen@mmc.edu Keywords: PTEN, SKP2, JARID1B, TRAF6, histone modifications, prostate cancer Received: September 02, 2014 Accepted: November 11, 2014 Published: December 23, 2014 ABSTRACT Aberrant elevation of JARID1B and histone H3 lysine 4 trimethylation (H3K4me3) is frequently observed in many diseases including prostate cancer (PCa), yet the mechanisms on the regulation of JARID1B and H3K4me3 through epigenetic alterations still remain poorly understood. Here we report that Skp2 modulates JARID1B and H3K4me3 levels in vitro in cultured cells and in vivo in mouse models. We demonstrated that Skp2 inactivation decreased H3K4me3 levels, along with a reduction of cell growth, cell migration and malignant transformation of Pten/Trp53 double null MEFs, and further restrained prostate tumorigenesis of Pten/Trp53 mutant mice. Mechanistically, Skp2 decreased the K63-linked ubiquitination of JARID1B by E3 ubiquitin ligase TRAF6, thus decreasing JARID1B demethylase activity and in turn increasing H3K4me3. In agreement, Skp2 deficiency resulted in an increase of JARID1B ubiquitination and in turn a reduction of H3K4me3, and induced senescence through JARID1B accumulation in nucleoli of PCa cells and prostate tumors of mice. Furthermore, we showed that the elevations of Skp2 and H3K4me3 contributed to castration-resistant prostate cancer (CRPC) in mice, and were positively correlated in human PCa specimens. Taken together, our findings reveal a novel network of SKP2-JARID1B, and targeting SKP2 and JARID1B may be a potential strategy for PCa control.

BACKGROUND The intervention of advanced prostate cancer (PCa) in patients has been commonly depending on androgen deprivation therapy. Despite of tremendous research efforts, however, molecular mechanisms on AR regulation remain poorly understood, particularly for castration resistant prostate cancer (CRPC). Targeting AR and associated factors is considered an effective strategy in PCa treatment. METHODS Human prostate cancer cells were used in this study. Manipulations of Skp2 expression were achieved by Skp2 shRNA/siRNA or overexpression of plasmids. Dual luciferase reporter assay was applied for AR activity assessment. Western blot, ubiquitination assay, immunoprecipitation, and immunofluorescence were applied to detect the proteins. RESULTS Our results demonstrated that Skp2 directly involves the regulation of AR expression through ubiquitination-mediated degradation. Skp2 interacted with AR protein in PCa cells, and enforced expression of Skp2 resulted in a decreased level and activity of AR. By contrast, Skp2 knockdown increased the protein accumulation and activity of AR. Importantly, changes of AR contributed by Skp2 led to subsequent alterations of PSA level in PCa cells. AR ubiquitination was significantly increased upon Skp2 overexpression but greatly reduced upon Skp2 knockdown. AR mutant at K847R abrogated Skp2-mediated ubiquitination of AR. NVP-BEZ235, a dual PI3K/mTOR inhibitor, remarkably inhibited Skp2 level with a striking elevation of AR. CONCLUSIONS The results indicate that Skp2 is an E3 ligase for proteasome-dependent AR degradation, and K847 on AR is the recognition site for Skp2-mediated ubiquitination. Our findings reveal an essential role of Skp2 in AR signaling. Prostate 74:421–432, 2014. © 2013 Wiley Periodicals, Inc.

Otoconia are biomineral particles of microscopic size essential for perception of gravity and maintenance of balance. Millions of older Americans are affected in their mobility, quality of life and in their health by progressive demineralization of otoconia. Currently, no effective means to prevent or counteract this process are available. Because of prohibitive anatomical and biological constraints, otoconial research is lagging far behind other systems such as bone and teeth. We have overcome these obstacles by generating otoconial matrix proteins by recombinant techniques. In the present study, we evaluated the effects of recombinant Otoconin 90 (OC90), the principal soluble matrix protein upon calcite crystal growth patterns in vitro. Our findings highlight multiple effects, including facilitation of nucleation, and inhibition of crystal growth in a concentration-dependent manner. Moreover, OC90 induces morphologic changes characteristic of native otoconia. OC90 is considerably less acidic than the prototypical invertebrate CaCO(3) -associated protein, but is nevertheless an effective modulator of calcite crystal growth. Based on homology modeling of the sPLA2-like domains of OC90, we propose that the lower density of acidic residues of the primary sequence is compensated by formation of major anionic surface clusters upon folding into tertiary conformation.

Myeloperoxidase (MPO), an enzyme derived from neutrophils, metabolically activates a wide range of carcinogens, whereas glutathione S-transferase M1 (GSTM1) detoxifies various electrophilic metabolites. A -463GA polymorphism in the promoter region of the MPO gene diminishes the expression of MPO and has been consistently shown to be associated with reduced risk of lung cancer in different ethnic populations. In our study, we have assessed the role of this polymorphism in lung cancer risk in a Chinese population. Genotypes of MPO and GSTM1 were determined by PCR-SSCP and multiplex PCR in 314 patients with lung cancer and 320 frequency-matched controls. The allele frequency for MPO -463A was found to be 0.155 in controls and 0.114 in cases. Subjects with the MPO -463GG genotype were at an increased risk of squamous cell carcinoma (SCC) of the lung compared to those having at least one -463A variant allele (adjusted odds ratio [OR] 2.35; 95% confidence interval [CI] 1.40–3.94). Stratified analysis suggested an interaction between heavy smoking (≥26 pack-years) and the MPO-463GG genotype. The adjusted OR of lung SCC for those having MPO-463GG genotype and smoked ≥26 pack-years was 20.50 (95% CI 5.58–75.33) compared to 6.22 (95% CI 1.72–22.47) for those smoked ≥26 pack-years but having at least one variant A allele (p = 0.023, test for homogeneity). This effect of the MPO polymorphism was not observed in lung adenocarcinoma. GSTM1 deletion was quite common in both controls (49.4%) and cases (50.3%) but was not associated with risk of lung cancer alone or in combination with the MPO polymorphism. Our results confirm the previous reports showing that the variant A allele of MPO has a protective effect against risk of lung cancer. © 2002 Wiley-Liss, Inc.

Quetiapine is a new atypical antipsychotic drug widely used in the treatment of schizophrenia and other psychotic disorders. This study examined the influence of quetiapine on the decrease of brain-derived neurotrophic factor (BDNF) expression, induced by chronic immobilization stress, in the hippocampus of the rat. Pretreatment with 10 mg/kg of quetiapine markedly attenuated the stress-induced decrease in levels of BDNF protein, as determined by Western blot analyses, and the reduction of BDNF immunoreactivity, in hippocampal pyramidal and dentate granular neurons. These results suggest that the chronic administration of quetiapine could be neuroprotective to hippocampal neurons in schizophrenia and this effect may be related to its antipsychotic effect in patients with schizophrenia.

Enhancer of zeste homolog 2 (EZH2) plays crucial roles on the development of cells and tissues as well as the progression of prostate cancer (PCa) and castration resistant prostate cancer (CRPC). EZH2 can upregulate and activate progenitor genes including androgen receptor (AR) and the target genes. However, the mechanisms by which EZH2 is regulated in PCa remain elusive. Literature reports that tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6) is essential for both normal tissue development and regulation of oncogenic signaling pathways in cancers. In this study, we reported that EZH2 is regulated by PTEN and SKP2 network in PCa. Specifically, we showed that the aberrant upregulation of EZH2 and histone H3 lysine 27 trimethylation (H3K27me3) in both Pten null mouse embryonic fibroblasts (MEFs) in vitro and prostate tumors of Pten null mice in vivo, as compared to the controls. EZH2 levels were negatively correlated with TRAF6 in human PCa cells upon SKP2 dysregulation. Immunofluorescence (IF) staining results showed a co-localization of EZH2 and TRAF6 in nucleus of PC3 cells, and co-immunoprecipitation (co-IP) analysis further confirmed a physical binding of EZH2 and TRAF6 in PC3 cells. Ectopic expression of TRAF6 promoted the K63-linked polyubiquitination of EZH2 to decrease EZH2 and H3K27me3 levels in PCa cells. Conversely, TRAF6 knockdown resulted in a reduction of EZH2 polyubiquitination with an increase of EZH2 and H3K27me3 levels in PCa cells. Furthermore, the catalytically dead mutant TRAF6 C70A abolished the TRAF6-mediated polyubiquitination of human EZH2 in vivo in 293T cells and in vitro in the recombinant human EZH2 protein, as compared with TRAF6 WT. Together, we report for the first time a novel mechanism on EZH2 ubiquitination and an important signaling network of SKP2-TRAF6-EZH2-H3K27me3 in PCa cells. Our findings provide valuable clues on the mechanism and efficiency of targeting EZH2 in PCa and CRPC . Note: This abstract was not presented at the meeting. Citation Format: Wenfu Lu, Shenji Lu, Bo Li, Yingqiu Xie, Michael G. Izban, Billy R. Ballard, Sandeep A. Sathyanarayana, Samuel E. Adunyah, Robert J. Matusik, Zhenbang Chen. EZH2 regulation through lysine 63 linked ubiquitination in prostate cancer [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 5326. doi:10.1158/1538-7445.AM2017-5326

Castration-resistant prostate cancer (PCa) (CRPC) is relapse after various forms of androgen ablation therapy and causes a major mortality in PCa patients, yet the mechanism remains poorly understood. Here, we report the nuclear form of mesenchymal epithelial transition factor (nMET) is essential for CRPC. Specifically, nMET is remarkably increased in human CRPC samples compared with naïve samples. Androgen deprivation induces endogenous nMET and promotes cell proliferation and stem-like cell self-renewal in androgen-nonresponsive PCa cells. Mechanistically, nMET activates SRY (sex determining region Y)-box9, β-catenin, and Nanog homeobox and promotes sphere formation in the absence of androgen stimulus. Combined treatment of MET and β-catenin enhances the inhibition of PCa cell growth. Importantly, MET accumulation is detected in nucleus of recurrent prostate tumors of castrated Pten/Trp53 null mice, whereas MET elevation is predominantly found in membrane of naïve tumors. Our findings reveal for the first time an essential role of nMET association with SOX9/β-catenin in CRPC in vitro and in vivo, highlighting that nuclear RTK activate cell reprogramming to drive recurrence, and targeting nMET would be a new avenue to treat recurrent cancers.

SLUG represses E-cadherin to promote epithelial-mesenchymal transition (EMT) in various cancers. Mechanisms that regulate SLUG/E-cadherin pathway remain poorly understood, especially during tumorigenesis in vivo. Here we report that p19(Arf) (p14(ARF) in human) stabilizes Slug to inhibit E-cadherin in prostate cancer mouse models. Inactivation of p19(Arf) reduces Slug levels, resulting in increased E-cadherin expression and delaying the onset and progression of prostate cancer in Pten/Trp53 double null mice. Mechanistically, p14(ARF) stabilizes SLUG through increased sumoylation at lysine residue 192. Importantly, levels of SLUG and p14(ARF) are positively correlated in human prostate cancer specimens. These data demonstrated that ARF modulates the SLUG/E-cadherin signaling axis for augmenting prostate tumorigenesis in vivo, revealing a novel paradigm where the oncogenic functions of SLUG require ARF to target E-cadherin in prostate cancer. Collectively, our findings further support that ARF has dual tumor suppressive/oncogenic roles in cancers in a context-dependent manner.

Prostate cancer (PCa) primarily depends on the dysregulations of androgen receptor (AR) signaling pathway for the initiation and growth as well as recurrence after chemotherapy [1]. Androgen deprivation therapy (ADT) effectively alleviates symptoms of the malignancy to arrest further growth of primary tumors or progression of metastasis in patients with advanced PCa. However, relapse occurs in many patients after a short period, and PCa cells eventually become insensitive to ADT - termed castration resistant prostate cancer (CRPC) [2, 3]. Tremendous advancements have been achieved to decipher the mechanisms on AR signaling, and the ubiquitination machinery contributes to PCa directly or indirectly by either promotion of AR transcriptional activity or degradation of AR protein levels. The recent report reveals that SKP2 regulates AR protein through ubiquitin-mediated proteasomal degradation, highlighting the role of SKP2 in AR signaling. Given the pivotal roles of AKT and SKP2 in cancers, the differential mechanisms of AR ubiquitination by various E3 ligases hold valuable significance and beneficial implications for PCa control.

Otoconia are formed embryonically and are instrumental in detecting linear acceleration and gravity. Degeneration and fragmentation of otoconia in elderly patients leads to imbalance resulting in higher frequency of falls that are positively correlated with the incidence of bone fractures and death. In this work we investigate the roles otoconial proteins Otolin-1 and Otoconin 90 (OC90) perform in the formation of otoconia. We demonstrate by rotary shadowing and atomic force microscopy (AFM) experiments that Otolin-1 forms homomeric protein complexes and self-assembled networks supporting the hypothesis that Otolin-1 serves as a scaffold protein of otoconia. Our calcium carbonate crystal growth data demonstrate that Otolin-1 and OC90 modulate in vitro calcite crystal morphology but neither protein is sufficient to produce the shape of otoconia. Coadministration of these proteins produces synergistic effects on crystal morphology that contribute to morphology resembling otoconia.

The intervention of advanced prostate cancer (PCa) in patients has been commonly depending on androgen deprivation therapy. Despite of tremendous research efforts, however, molecular mechanisms on AR regulation remain poorly understood, particularly for castration resistant prostate cancer (CRPC). Targeting AR and associated factors is considered an effective strategy in PCa treatment.Human prostate cancer cells were used in this study. Manipulations of Skp2 expression were achieved by Skp2 shRNA/siRNA or overexpression of plasmids. Dual luciferase reporter assay was applied for AR activity assessment. Western blot, ubiquitination assay, immunoprecipitation, and immunofluorescence were applied to detect the proteins.Our results demonstrated that Skp2 directly involves the regulation of AR expression through ubiquitination-mediated degradation. Skp2 interacted with AR protein in PCa cells, and enforced expression of Skp2 resulted in a decreased level and activity of AR. By contrast, Skp2 knockdown increased the protein accumulation and activity of AR. Importantly, changes of AR contributed by Skp2 led to subsequent alterations of PSA level in PCa cells. AR ubiquitination was significantly increased upon Skp2 overexpression but greatly reduced upon Skp2 knockdown. AR mutant at K847R abrogated Skp2-mediated ubiquitination of AR. NVP-BEZ235, a dual PI3K/mTOR inhibitor, remarkably inhibited Skp2 level with a striking elevation of AR.The results indicate that Skp2 is an E3 ligase for proteasome-dependent AR degradation, and K847 on AR is the recognition site for Skp2-mediated ubiquitination. Our findings reveal an essential role of Skp2 in AR signaling.

Androgen receptor (AR) signaling is essential for prostate cancer (PCa) development in humans. The initiation of prostate malignancy and progression to a castration-resistant stage are largely contributed by the modulation of AR activity through its coregulatory proteins. We and others previously reported that p14 alternative reading frame (ARF) expression is positively correlated with the disease progression and severity of PCa. Here, we provide evidence that p14ARF physically interacts with AR and functions as an AR corespressor in both an androgen-dependent and androgen-independent manner. Endogenous ARF (p14ARF in human and p19ARF in mouse) and AR colocalize in both human PCa cells in vitro and PCa tissues of mouse and human in vivo. Overexpression of p14ARF in PCa cells significantly attenuates the activities of androgen response region (ARR2)-probasin and prostate-specific antigen (PSA) promoters. The forced expression of p14ARF in cells resulted in a suppression of PSA and NK transcription factor locus 1 (NKX3.1) expression. Conversely, knockdown of endogenous p14ARF in human PCa cells with short hairpin RNA enhanced AR transactivation activities in a dose-dependent and p53-independent manner. Furthermore, we demonstrated that p14ARF binds to both the N-terminal domain and the ligand-binding domain of AR, and the human double minute 2 (HDM2)-binding motif of p14ARF is required for the interaction of p14ARF and AR proteins. p14ARF perturbs the androgen-induced interaction between the N terminus and C terminus of AR. Most importantly, we observed that the expression of PSA is reversely correlated with p14ARF in human prostate tissues. Taken together, our results reveal a novel function of ARF in modulation of AR transactivation in PCa.

Co-option of the enzyme secretory phospholipase A2 (sPLA2) and adoption of tertiary conformation are essential factors in the multifunctionality of otoconin 90 (OC90) and homologous modulators.To present results of in vitro studies of recombinant otoconial proteins for the understanding of current concepts of biomolecular mechanisms controlling otoconial mineralization.In vitro characterization of recombinant otoconial proteins with respect to crystal growth parameters and solution state behavior. Evaluation by HR-SEM, micro-Raman, circular dichroism, in combination with molecular modeling of individual domains and whole OC90.Polymorph selection: recombinant otoconin 22 (rOC22) in vitro selects calcite rather than aragonite, expression of which requires association with an insoluble scaffold most likely provided by Otolin. Alternate folding of rOC22 results in formation of vaterite, the polymorph of primitive fish otoconia and of diseased human otoconia (e.g. Potter's syndrome). Molecular models of OC90 exhibit a surface of uniform negative electrostatic potential, enabling localized supersaturation. We propose that OC90 interacts with Otolin in formation of iso-oriented columns of nano-crystallites, which should ultimately result in assembly of the complex mosaic of native otoconia.

p53 regulates an array of target genes, which mediates p53 tumor suppression by inducing cell cycle arrest, apoptosis, and cell survival. G protein–coupled receptors belong to a superfamily of cell surface molecules and are known to regulate cell proliferation, migration, and survival. Here, we found that G protein–coupled receptor 87 (GPR87) was up-regulated by p53 and by DNA damage in a p53-dependent manner. We also found that p53 directly regulated GPR87 potentially via a p53-responsive element in the GPR87 gene. To investigate the role of GPR87 in the p53 pathway, we generated multiple RKO and MCF7 cell lines in that GPR87 can be inducibly overexpressed or knocked down by a tetracycline-inducible system. We found that overexpression of GPR87 had little effect on cell growth. However, GPR87 knockdown sensitized cancer cells to DNA damage–induced growth suppression via enhanced p53 stabilization and activation. Importantly, the prosurvival activity of GPR87 can be reversed by knockdown of p53. Together, our results suggested that GPR87 is essential for p53-dependent cell survival in response to DNA damage. Thus, due to its expression on the cell surface and its role in cell survival, GPR87 may be explored as a novel therapeutic target for cancer treatment and prevention. [Cancer Res 2009;69(15):6049–56]

Aberrant elevation of JARID1B and histone H3 Lys4 trimethylations (H3K4me3) is frequently observed in many diseases including prostate cancer (PCa), yet the mechanisms on the regulations of JARID1B and H3K4me3 through epigenetic modifications still remain poorly understood. In this study we performed immunohistochemistry staining, immunofluorescence imaging, immunoprecipitation, shRNA and Western blotting analysis in mouse embryonic fibroblasts (MEFs), mouse models, and cultured human prostate cancer cells. As a result, we discovered that SKP2 modulates JARID1B and H3K4me3 levels in vitro in PTEN null prostate cancer cells and in vivo in Pten/Trp53 mouse models. We demonstrated that levels of SKP2, JARID1B and H3K4me3 are strikingly elevated in vitro and in vivo when both PTEN and P53 are inactivated. Importantly, SKP2 inactivation resulted in a reduction of cell growth, cell migration and malignant transformation of Pten/Trp53 double null MEFs, and further restrained prostate tumorigenesis of Pten/Trp53 mutant mice. Mechanistically, JARID1B is ubiquitinated by E3 ligase TRAF6 through the K63-linkage in prostate cancer cells. Interestingly, SKP2 contributes to JARID1B ubiquitination machinery as a non-E3 ligase regulator by decreasing TRAF6-mediated ubiquitination of JARID1B. SKP2 deficiency resulted in an increase of JARID1B ubiquitination and in turn a reduction of H3K4me3, and induced senescence through JARID1B accumulation in nucleoli of PCa cells and prostate tumors of mice. Furthermore, we showed that the aberrant levels of SKP2, JARID1B, and H3K4me3 are associated with malignant features of castration-resistant prostate cancer (CRPC) in mice. Overall, our findings reveal a novel network of SKP2- JARID1B, and targeting SKP2 and JARID1B may be a potential strategy for PCa control. Citation Format: Wenfu Lu, Shenji Liu, Bo Li, Yingqiu Xie, Christine Adhiambo, Qing Yang, Billy R. Ballard, Keiichi I. Nakayama, Robert J. Matusik, Zhenbang Chen. K63-linked JARID1B ubiquitination by TRAF6 contributes to aberrant elevation of JARID1B in prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 90. doi:10.1158/1538-7445.AM2015-90

The development of esophageal squamous cell carcinoma (ESCC) has been linked to exposure to carcinogens such as nitrosamines that cause various alkyl DNA damages and O6-methylguanine-DNA methyltransferase (MGMT) is a primary defence against alkylation-induced mutagenesis and carcinogenesis. This study was to investigate the role of inactivation of MGMT by promoter hypermethylation and its relation to p53 mutations in ESCC. Methylation of MGMT promoter was determined by methylation-specific polymerase chain reaction in 119 ESCC specimens, 22 corresponding tissue samples adjacent to the tumors, and 21 normal epithelial specimens of the esophagus. The levels of MGMT protein in ESCC with methylated or unmethylated MGMT were analyzed by quantitative immunohistochemistry. Mutations of p53 in 119 ESCC were detected by denaturing high-performance liquid chromatography and sequencing. We found that all 21 normal esophageal tissues had unmethylated MGMT; however, among 119 ESCC, 46 (38.7%) had hypermethylated MGMT. This epigenetic change also occurred in some normal tissues adjacent to the tumors. The level of MGMT protein in MGMT-methylated ESCC was significantly lower than that in MGMT-unmethylated ESCC, whereas great inter-individual variation and poor expression was also observed among MGMT-unmethylated ESCC. Fifty-one percent (61/119) ESCC showed p53 mutations but the distribution of the mutations did not differ significantly between MGMT-methylated ESCC (44%) and MGMT-unmethylated ESCC (56.2%; P = 0.18). MGMT promoter hypermethylation was neither associated with overall G:C to A:T mutations nor associated with this type of mutations in non-CpG dinucleotides in p53. Our results demonstrate that inactivation of MGMT by aberrant promoter methylation is a frequent molecular event in ESCC. This epigenetic alteration is an important, but may not be the sole, mechanism leading to the impaired expression of MGMT. Aberrant MGMT methylation seemed not to be associated with overall frequency and spectrum of p53 mutations in ESCC.

Variation in DNA repair capacity, which is believed to be largely determined by genetic traits, is linked to risk of certain cancers. The Asp312Asn and Lys751Gln polymorphisms in the xeroderma pigmentosum complementary group D (XPD) gene may alter DNA repair capacity. We thus examined the hypothesis that these 2 XPD polymorphisms are associated with risk of lung cancer via a large hospital-based, case-control study among Chinese. The study subjects consisted of 1,006 patients with primary lung cancer and 1,020 age- and sex-matched population controls. XPD genotypes were determined using PCR-RFLP techniques, and the associations between genotypes and risk of lung cancer were estimated by odds ratios (ORs) and their 95% confidence intervals (CIs) calculated by unconditional logistic regression. Subjects homozygous for the 312Asn/Asn genotype had an increased risk of lung cancer (adjusted OR = 10.33, 95% CI = 1.29-82.50) compared with subjects homozygous for the 312Asp/Asp genotype. The 751Gln/Gln genotype was also associated with increased risk for the cancer compared with the 751Lys/Lys genotype (adjusted OR = 2.71, 95% CI = 1.01-7.24). Stratification analysis revealed that the increased risk was mainly confined to lung squamous cell carcinoma, with the ORs being 20.50 (95% CI = 2.25-179.05) for the 312Asn/Asn genotype and 4.24 (95% CI = 1.34-13.38) for the 751Gln/Gln genotype, respectively. Haplotype analysis with the 2 polymorphisms suggested these polymorphisms might be in linkage disequilibrium with a different causative locus or act together with other functional variants in or close to the XPD locus.

To examine the relationship between susceptibility to lung cancer among Chinese and genetic polymorphism at nucleotide -463 (G/A) in myeloperoxidase (MPO), an enzyme found in lysosomes of phagocytes and involved in the formation of hydroxyl radicals and activation of various smoking-related carcinogens.The association of this polymorphism with lung cancer in a hospital-based case-control study of 314 patients and 320 age- and sex-matched controls was tested. The MPO genotypes were determined by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP).The allele frequency for MPO-463A was found to be 11.0% for controls, compared with 15.0% for patients. Multivariate analysis showed an increased risk for overall lung cancer in subjects having GG genotype (OR, 1.7; 95% CI, 1.2 - 2.5), however, the elevated risk was limited to squamous cell carcinoma (OR, 2.4; 95% CI, 1.4 - 3.9; n = 177) but not to adenocarcinoma (OR, 1.3; 95% CI, 0.8 - 2.1; n = 137). In addition, the risky effect of the GG genotype on squamous cell carcinoma of the lung was evident only in the smokers and those who smoked/= 26 pack-years (OR, 20.5; 95% CI, 5.6 - 75.3) as compared with GA and AA genotypes (OR, 6.2; 95% CI, 1.7 - 22.5) but not in the nonsmokers or those who smoked26 pack-years.Our data support the hypothesis that -463A polymorphism in the MPO gene may reduce the susceptibility to lung cancer in the Chinese.

Methylenetetrahydrofolate reductase (MTHFR) plays a centralrole in converting folate to methyl donor for DNA methylation, an epigenetic modification known to be dysregulated in carcinogenesis. Our previous study revealed that MTHFR polymorphisms contribute to a great risk of esophageal cancer in a Chinese population. This case-control study was to examine the association between MTHFR polymorphisms and gastric cardia adenocarcinoma (GCA), which is also prevalent in high-risk areas of esophageal cancer and thus may share common etiological factors with esophageal cancer in this population. The study subjects were 217 patients with GCA and 468 population controls matched on sex and age. The MTHFR C677T and A1298C genotypes were detected by a PCR-based RFLP assay. It was found that subjects with the MTHFR 677TT variant genotype had a 2-fold increased risk of GCA (odds ratio, 2.04; 95% confidence interval, 1.28-3.26). Moreover, a significantly elevated risk was also seen among the MTHFR 677CT heterozygotes (odds ratio, 1.56; 95% confidence interval, 1.03-2.36). The MTHFR A1298C polymorphism had no effect on risk of GCA. These findings are generally consistent with our initial observation for esophageal cancer and suggest that the MTHFR genotype may be a determinant of GCA among this at-risk Chinese population.

To explore the relationship between genetic polymorphisms in methylenetetrahydrofolate reductase (MTHFR), a central enzyme in folate metabolism that affects DNA methylation and synthesis, and the risk of adenocarcinoma of the gastric cardia (AGC).Genomic DNA was isolated from the peripheral lymphocytes of 205 patients with AGC and 360 age-and-sex-matched control subjects. Polymerase chain reaction and restriction fragment length polymorphism were used to examine the polymorphisms of MTHFR genes C6677T and A1298C and the relation between these genotypes and risk of AGC was analyzed.The genotype frequency of MTHFR C677CC was 35.0% among the controls and was 20.5% among the AGC patients; the genotype frequency of 677CT was 47.8% among the controls and 50.7% among the AGC patients, and the genotype frequency of 677TT was 17.2% among the controls and 28.8% among the AGC patients % (chi(2) = 17.63, P = 0.0002). The individuals with 677CT genotype or 677TT genotype had a 1.76-fold [95% confidence interval (CI), 1.13 - 2.76] or 2.97-fold (95% CI, 1.75 - 5.05) increased risk of developing AGC compared with those who had 677CC genotype. The distribution of MTHFR A1298C genotype was not significantly different between the two groups (chi(2) = 0.08, P = 0.96). The value of odd ratio (OR) in the individuals with 677CT//1298AA genotype (95% CI, 1.48 approximately 6.48), and the value of OR in the individuals with 677CT/1298CC genotype was 17.00 (95% CI, 1.26 approximately infinity; P = 0.042).Single nucleotide polymorphism in the MTHFR gene is a risk factor of carcinogenesis of AGC in the Chinese population. Although the MTHFR A1298C polymorphism was not associated with AGC, The risk of developing AGC in the individuals with MTHFR677CT/1298CC was 17 times higher than that in the individuals with MTHFR677CC/1298AA, thus showing that a joint effect exists between the MTHFR 677 and 1298 polymorphisms on the risk of AGC.

Esophageal squamous cell carcinoma (ESCC), which is prevalent in China, is believed to be induced by environmental carcinogens. Accumulating evidence has shown that individual variation in DNA repair capacity resulting from genetic polymorphism influences risk of environmental carcinogenesis. We therefore investigated the associations between genetic polymorphisms in the DNA repair genes XRCC1 (Arg194Trp and Arg399Gln) and XPD (Asp312Asn and Lys751Gln) and risk of ESCC in an at-risk Chinese population. Genotypes were determined by a PCR-based approach in 433 patients with ESCC and 524 frequency-matched normal controls. We found that individuals with Trp/Trp genotype at XRCC1 Arg194Trp site had a 2-fold increased risk of this disease compared to Arg/Arg genotype (adjusted OR = 1.98; 95% CI 1.26-3.12). Furthermore, when compared to Arg/Arg and Arg/Trp genotype combined, homozygote for Trp/Trp genotype significantly increased the risk of developing ESCC, with the adjusted OR being 2.07 (95% CI 1.34-3.20). However, the XRCC1 Arg399Gln polymorphism was not significantly associated with risk of ESCC, with the adjusted OR being 0.87 (95% CI 0.55-1.37). Neither Asp312Asn nor Lys751Gln polymorphisms in the XPD gene influenced risk of ESCC in our study. These findings suggest that DNA repair gene XRCC1 but not XPD might play a role in esophageal carcinogenesis and might represent a genetic determinant in the development of the cancer.

Purpose: Prostate Cancer (PCa) is the second leading cause of cancer-related deaths in American men. The morbidity and mortality to PCa are 2.39-fold higher in African American men as compared to Caucasian males. Aberrant overexpression of SKP2 is frequently found in human cancers including prostate cancer, but little is known about pharmacological compounds targeting Skp2 activity. This study investigated inhibitory effects of curcumin (diferuloylmethane) on malignant features of prostate cancer cells in vitro and xenograft mouse models in vivo, and mechanistically on Skp2 level. Design Methods: Prostate cancer cells (PC3) and mouse prostate cancers cells (MPC3) in culture were treated with curcumin solution for indicated periods of time in vitro. Quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) and Western blot analyses were used to determine mRNA and protein expression of Skp2 in cancer cells. Cell proliferation, colony formation, and invasion assay were compared between curcumin-treated and control cells. Mouse prostate cancer cells were injected into immunodeficient mice followed with curcumin administration (200mg/kg/day). Tumorigenesis was analyzed in curcumin-treated and control mice after 20 days. Results: Curcumin treatment of PC3 cells resulted in a significant reduction of cell proliferation, soft agar transformation and invasion as compared to controls. Curcumin treatment led to a decrease of Skp2 level through a reduction of Skp2 mRNA and a shortened half-life of Skp2 protein. As expected, p27 protein, a major downstream target of Skp2, was increased upon curcumin treatment, whereas c-Myc protein level was not affected by curcumin treatment in prostate cancer cells. MPC3 cells, administered into immunodeficient mice via tail vein or orthotopic implantation, developed lung metastasis or enlarged prostate tumors, respectively. Importantly, cancer progression was significantly suppressed by curcumin treatment. A reduced level of Skp2 protein and induction of cell senescence were detected in tumors of curcumin-treated mice, but not in tumors of control mice. Conclusion: Our results indicated that curcumin suppresses both the tumor growth in mouse prostates and distant metastasis of prostate cancer cells in lung by inhibiting Skp2 level. Citation Format: Yuanying Gong, Wenfu Lu, Yingqiu Xie, Qing Yang, Robert J. Matusik, Zhenbang Chen. Curcumin suppresses the growth and metastasis of prostate cancer cells by downregulation of SKP2. [abstract]. In: Proceedings of the Fifth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2012 Oct 27-30; San Diego, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(10 Suppl):Abstract nr B23.