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Find video protocols related to scientific articles indexed in Pubmed.
Abiraterone Treatment in Castration-Resistant Prostate Cancer Selects for Progesterone Responsive Mutant Androgen Receptors.
Clin. Cancer Res.
PUBLISHED: 10-17-2014
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Purpose:The CYP17A1 inhibitor abiraterone markedly reduces androgen precursors and is thereby effective in castration-resistant prostate cancer (CRPC). However, abiraterone increases progesterone, which can activate certain mutant androgen receptors (ARs) identified previously in flutamide-resistant tumors. Therefore, we sought to determine if CYP17A1 inhibitor treatment selects for progesterone activated mutant ARs. Experimental Design:AR was examined by targeted sequencing in metastatic tumor biopsies from 18 CRPC patients who were progressing on a CYP17A1 inhibitor (17 on abiraterone, 1 on ketoconazole), alone or in combination with dutasteride, and by whole exome sequencing in residual tumor in one patient treated with neoadjuvant leuprolide plus abiraterone. Results:The progesterone-activated T878A mutant AR was present at high allele frequency in 3 of the 18 CRPC cases. It was also present in one focus of resistant tumor in the neoadjuvant treated patient, but not in a second clonally related resistant focus which instead had lost one copy of PTEN and both copies of CHD1. The T878A mutation appeared to be less common in the subset of CRPC patients treated with abiraterone plus dutasteride, and transfection studies showed that dutasteride was a more potent direct antagonist of the T878A versus the wildtype AR. Conclusions:These findings indicate that selection for tumor cells expressing progesterone-activated mutant ARs is a mechanism of resistance to CYP17A1 inhibition.
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Intense Androgen-Deprivation Therapy With Abiraterone Acetate Plus Leuprolide Acetate in Patients With Localized High-Risk Prostate Cancer: Results of a Randomized Phase II Neoadjuvant Study.
J. Clin. Oncol.
PUBLISHED: 10-13-2014
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Cure rates for localized high-risk prostate cancers (PCa) and some intermediate-risk PCa are frequently suboptimal with local therapy. Outcomes are improved by concomitant androgen-deprivation therapy (ADT) with radiation therapy, but not by concomitant ADT with surgery. Luteinizing hormone-releasing hormone agonist (LHRHa; leuprolide acetate) does not reduce serum androgens as effectively as abiraterone acetate (AA), a prodrug of abiraterone, a CYP17 inhibitor that lowers serum testosterone (< 1 ng/dL) and improves survival in metastatic PCa. The possibility that greater androgen suppression in patients with localized high-risk PCa will result in improved clinical outcomes makes paramount the reassessment of neoadjuvant ADT with more robust androgen suppression.
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Whole Transcriptome Sequencing Reveals Extensive Unspliced mRNA in Metastatic Castration-Resistant Prostate Cancer.
Mol. Cancer Res.
PUBLISHED: 09-04-2014
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Men with metastatic prostate cancer (PCa) who are treated with androgen deprivation therapies (ADT) usually relapse within 2-3 years with disease that is termed castration-resistant prostate cancer (CRPC). To identify the mechanism that drives these advanced tumors, paired-end RNA-sequencing (RNA-seq) was performed on a panel of CRPC bone marrow biopsy specimens. From this genome-wide approach, mutations were found in a series of genes with PCa relevance including: AR, NCOR1, KDM3A, KDM4A, CHD1, SETD5, SETD7, INPP4B, RASGRP3, RASA1, TP53BP1 and CDH1, and a novel SND1:BRAF gene fusion. Amongst the most highly-expressed transcripts were ten non-coding RNAs (ncRNAs), including MALAT1 and PABPC1, which are involved in RNA processing. Notably, a high percentage of sequence reads mapped to introns, which were determined to be the result of incomplete splicing at canonical splice junctions. Using quantitative PCR (qPCR) a series of genes (AR, KLK2, KLK3, STEAP2, CPSF6, and CDK19) were confirmed to have a greater proportion of unspliced RNA in CRPC specimens than in normal prostate epithelium, untreated primary PCa, and cultured PCa cells. This inefficient coupling of transcription and mRNA splicing suggests an overall increase in transcription or defect in splicing. Implications: Inefficient splicing in advanced prostate cancer provides a selective advantage through effects on micro-RNA networks, but may render tumors vulnerable to agents that suppress rate-limiting steps in splicing.
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The cistrome and gene signature of androgen receptor splice variants in castration-resistant prostate cancer cells.
J. Urol.
PUBLISHED: 08-14-2014
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Spliced variant forms of androgen receptor (AR-Vs) have been identified recently in castration-resistant prostate cancer (CRPC) cell lines and clinical samples. We identified the cistrome and gene signature of AR-Vs in CRPC cell lines and determined the clinical significance of AR-Vs regulated genes.
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Galeterone prevents androgen receptor binding to chromatin and enhances degradation of mutant androgen receptor.
Clin. Cancer Res.
PUBLISHED: 05-29-2014
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Galeterone inhibits the enzyme CYP17A1 and is currently in phase II clinical trials for castration-resistant prostate cancer (CRPC). Galeterone is also a direct androgen receptor (AR) antagonist and may enhance AR degradation. This study was undertaken to determine the molecular basis for AR effects and their therapeutic potential.
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PTEN-deficient tumors depend on AKT2 for maintenance and survival.
Cancer Discov
PUBLISHED: 05-16-2014
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Loss of PTEN is a common event in many cancers and leads to hyperactivation of the PI3K-AKT signaling pathway. The mechanisms by which AKT isoforms mediate signaling to phenotypes associated with PTEN inactivation in cancer have not been defined. Here, we show that AKT2 is exclusively required for PTEN-deficient prostate tumor spheroid maintenance, whereas AKT1 is dispensable. shRNA silencing of AKT2 but not AKT1 promotes regression of prostate cancer xenografts. Mechanistically, we show that AKT2 silencing upregulates p21 and the proapoptotic protein BAX and downregulates the insulin-like growth factor receptor-1. We also show that p21 is an effector of AKT2 in mediating prostate tumor maintenance. Moreover, AKT2 is also exclusively required for the maintenance and survival of other PTEN-deficient solid tumors, including breast cancer and glioblastoma. These findings identify a specific function for AKT2 in mediating survival of PTEN-deficient tumors and provide a rationale for developing therapeutics targeting AKT2.
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Enhancer RNAs participate in androgen receptor-driven looping that selectively enhances gene activation.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 04-28-2014
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The androgen receptor (AR) is a key factor that regulates the behavior and fate of prostate cancer cells. The AR-regulated network is activated when AR binds enhancer elements and modulates specific enhancer-promoter looping. Kallikrein-related peptidase 3 (KLK3), which codes for prostate-specific antigen (PSA), is a well-known AR-regulated gene and its upstream enhancers produce bidirectional enhancer RNAs (eRNAs), termed KLK3e. Here, we demonstrate that KLK3e facilitates the spatial interaction of the KLK3 enhancer and the KLK2 promoter and enhances long-distance KLK2 transcriptional activation. KLK3e carries the core enhancer element derived from the androgen response element III (ARE III), which is required for the interaction of AR and Mediator 1 (Med1). Furthermore, we show that KLK3e processes RNA-dependent enhancer activity depending on the integrity of core enhancer elements. The transcription of KLK3e was detectable and its expression is significantly correlated with KLK3 (R(2) = 0.6213, P < 5 × 10(-11)) and KLK2 (R(2) = 0.5893, P < 5 × 10(-10)) in human prostate tissues. Interestingly, RNAi silencing of KLK3e resulted in a modest negative effect on prostate cancer cell proliferation. Accordingly, we report that an androgen-induced eRNA scaffolds the AR-associated protein complex that modulates chromosomal architecture and selectively enhances AR-dependent gene expression.
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Rapid induction of androgen receptor splice variants by androgen deprivation in prostate cancer.
Clin. Cancer Res.
PUBLISHED: 01-21-2014
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Mechanisms mediating androgen receptor (AR) reactivation in prostate cancer that progresses after castration (castration-resistant prostate cancer; CRPC) and subsequent treatment with abiraterone (CYP17A1 inhibitor that further suppresses androgen synthesis) remain unclear.
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Targeted Androgen Pathway Suppression in Localized Prostate Cancer: A Pilot Study.
J. Clin. Oncol.
PUBLISHED: 12-09-2013
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Ligand-mediated activation of the androgen receptor (AR) is critical for prostate cancer (PCa) survival and proliferation. The failure to completely ablate tissue androgens may limit suppression of PCa growth. We evaluated combinations of CYP17A and 5-?-reductase inhibitors for reducing prostate androgen levels, AR signaling, and PCa volumes.
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Castration-resistant prostate cancer: Adaptive responses in the androgen axis.
Cancer Treat. Rev.
PUBLISHED: 07-01-2013
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The androgen signaling axis in prostate cancer is associated with multiple adaptive mechanisms in response to castration. Herein we review these adaptations with an emphasis on recent molecular insights into the growth and development of castration resistant prostate cancer (CRPC). Alterations include both conventional and novel intracrine androgen synthesis pathways and androgen transport as well as androgen receptor (AR) overexpression, mutation, and splice variation. Each of these underlying mechanisms are potentially linked to post-castration growth, especially after treatment with newer hormonal agents such as abiraterone and enzalutamide. Post-translational AR modifications are well documented and these can affect receptor activity, stability, localization, and interaction with other proteins. Changes in recruitment of androgen receptor associated co-activators/repressors and a distinct AR-induced transcriptional program can dramatically alter proliferation, invasion, and metastasis in a ligand and context-dependent manner. Numerous previously uncharacterized non-coding RNAs, some of which are androgen regulated, may also have important biological function in this disease. Taken together, the view of CRPC has changed dramatically in the last several years. This has occurred not only within the setting of multiple treatment paradigm changes, but also as a multiplicity of potential molecular mechanisms underlying this disease state have been explored and discovered.
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Tyrosine kinase BMX phosphorylates phosphotyrosine-primed motif mediating the activation of multiple receptor tyrosine kinases.
Sci Signal
PUBLISHED: 05-30-2013
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The nonreceptor tyrosine kinase BMX (bone marrow tyrosine kinase gene on chromosome X) is abundant in various cell types and activated downstream of phosphatidylinositol-3 kinase (PI3K) and the kinase Src, but its substrates are unknown. Positional scanning peptide library screening revealed a marked preference for a priming phosphorylated tyrosine (pY) in the -1 position, indicating that BMX substrates may include multiple tyrosine kinases that are fully activated by pYpY sites in the kinase domain. BMX phosphorylated focal adhesion kinase (FAK) at Tyr??? subsequent to its Src-mediated phosphorylation at Tyr???. Loss of BMX by RNA interference or by genetic deletion in mouse embryonic fibroblasts (MEFs) markedly impaired FAK activity. Phosphorylation of the insulin receptor in the kinase domain at Tyr¹¹?? and Tyr¹¹??, as well as Tyr¹¹??, and downstream phosphorylation of the kinase AKT at Thr³?? were similarly impaired by BMX deficiency. However, insulin-induced phosphorylation of AKT at Ser??³ was not impaired in Bmx knockout MEFs or liver tissue from Bmx knockout mice, which also showed increased insulin-stimulated glucose uptake, possibly because of decreased abundance of the phosphatase PHLPP (PH domain leucine-rich repeat protein phosphatase). Thus, by identifying the pYpY motif as a substrate for BMX, our findings suggest that BMX functions as a central regulator among multiple signaling pathways mediated by tyrosine kinases.
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Discovery of a Selective Irreversible BMX Inhibitor for Prostate Cancer.
ACS Chem. Biol.
PUBLISHED: 04-26-2013
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BMX is a member of the TEC family of nonreceptor tyrosine kinases. We have used structure-based drug design in conjunction with kinome profiling to develop a potent, selective, and irreversible BMX kinase inhibitor, BMX-IN-1, which covalently modifies Cys496. BMX-IN-1 inhibits the proliferation of Tel-BMX-transformed Ba/F3 cells at two digit nanomolar concentrations but requires single digit micromolar concentrations to inhibit the proliferation of prostate cancer cell lines. Using a combinatorial kinase inhibitor screening strategy, we discovered that the allosteric Akt inhibitor, MK2206, is able to potentiate BMX inhibitors antiproliferation efficacy against prostate cancer cells.
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ERG induces androgen receptor-mediated regulation of SOX9 in prostate cancer.
J. Clin. Invest.
PUBLISHED: 02-15-2013
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Fusion of the androgen receptor-regulated (AR-regulated) TMPRSS2 gene with ERG in prostate cancer (PCa) causes androgen-stimulated overexpression of ERG, an ETS transcription factor, but critical downstream effectors of ERG-mediating PCa development remain to be established. Expression of the SOX9 transcription factor correlated with TMPRSS2:ERG fusion in 3 independent PCa cohorts, and ERG-dependent expression of SOX9 was confirmed by RNAi in the fusion-positive VCaP cell line. SOX9 has been shown to mediate ductal morphogenesis in fetal prostate and maintain stem/progenitor cell pools in multiple adult tissues, and has also been linked to PCa and other cancers. SOX9 overexpression resulted in neoplasia in murine prostate and stimulated tumor invasion, similarly to ERG. Moreover, SOX9 depletion in VCaP cells markedly impaired invasion and growth in vitro and in vivo, establishing SOX9 as a critical downstream effector of ERG. Finally, we found that ERG regulated SOX9 indirectly by opening a cryptic AR-regulated enhancer in the SOX9 gene. Together, these results demonstrate that ERG redirects AR to a set of genes including SOX9 that are not normally androgen stimulated, and identify SOX9 as a critical downstream effector of ERG in TMPRSS2:ERG fusion-positive PCa.
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Development, validation and application of a stable isotope dilution liquid chromatography electrospray ionization/selected reaction monitoring/mass spectrometry (SID-LC/ESI/SRM/MS) method for quantification of keto-androgens in human serum.
J. Steroid Biochem. Mol. Biol.
PUBLISHED: 01-19-2013
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Prostate cancer is the most frequently diagnosed form of cancer in males in the United States. The disease is androgen driven and the use of orchiectomy or chemical castration, known as androgen deprivation therapy (ADT) has been employed for the treatment of advanced prostate cancer for over 70 years. Agents such as GnRH agonists and non-steroidal androgen receptor antagonists are routinely used in the clinic, but eventually relapse occurs due to the emergence of castration-resistant prostate cancer. With the appreciation that androgen signaling still persists in these patients and the development of new therapies such as abiraterone and enzalutamide that further suppresses androgen synthesis or signaling, there is a renewed need for sensitive and specific methods to quantify androgen precursor and metabolite levels to assess drug efficacy. We describe the development, validation and application of a stable isotope dilution liquid chromatography electrospray ionization selected reaction monitoring mass spectrometry (SID-LC/ESI/SRM/MS) method for quantification of serum keto-androgens and their sulfate and glucuronide conjugates using Girard-T oxime derivatives. The method is robust down to 0.2-4pg on column, depending on the androgen metabolite quantified, and can also quantify dehydroepiandrosterone sulfate (DHEA-S) in only 1?L of serum. The clinical utility of this method was demonstrated by analyzing serum androgens from patients enrolled in a clinical trial assessing combinations of pharmacological agents to maximally suppress gonadal and adrenal androgens (Targeted Androgen Pathway Suppression, TAPS clinical trial). The method was validated by correlating the results obtained with a hydroxylamine derivatization procedure coupled with tandem mass spectrometry using selected reaction monitoring that was conducted in an independent laboratory.
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SOX9 regulates low density lipoprotein receptor-related protein 6 (LRP6) and T-cell factor 4 (TCF4) expression and Wnt/?-catenin activation in breast cancer.
J. Biol. Chem.
PUBLISHED: 01-10-2013
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Gene expression profiling has identified breast cancer (BCa) subtypes, including an aggressive basal-like (BL) subtype. The molecular signals underlying the behavior observed in BL-BCa group are largely unknown, although recent results indicate a prevalent increase in Wnt/?-catenin activity. Our immunohistochemistry study confirmed that SOX9, one of the BL-BCa signature genes, was expressed by most BL-BCa, and its expression correlated with indicators of poor prognosis. Importantly, BCa gene expression profiling strongly associated SOX9 with the expression of Wnt/?-catenin pathway components, LRP6 and TCF4. In cancer cell lines, SOX9 silencing reduced cell proliferation and invasion, LRP6 and TCF4 transcription, and decreased Wnt/?-catenin activation. SOX9 expression was also increased by Wnt, indicating that SOX9 is at the center of a positive feedback loop that enhances Wnt/?-catenin signaling. Consistently, SOX9 overexpression in BCa cell lines and transgenic SOX9 expression in breast epithelium caused increased LRP6 and TCF4 expression and Wnt/?-catenin activation. These results identify SOX9-mediated Wnt/?-catenin activation as one of the molecular mechanisms underlying aberrant Wnt/?-catenin activity in BCa, especially in the BL-BCa subgroup.
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Androgen Receptor Enhances p27 Degradation in Prostate Cancer Cells through Rapid and Selective TORC2 Activation.
J. Biol. Chem.
PUBLISHED: 12-02-2011
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Androgen receptor (AR) plays a central role in prostate cancer (PCa) growth, with androgen deprivation or AR down-regulation causing cell-cycle arrest and accumulation of the p27 cyclin-dependent kinase inhibitor. The molecular basis for this AR regulation of cell-cycle progression remains unclear. Here we demonstrate that androgen can rapidly reduce p27 protein in PCa cells by increasing its proteasome-mediated degradation. This rapid androgen-stimulated p27 degradation was mediated by AKT through the phosphorylation of p27 T157. Significantly, androgen increased TORC2-mediated AKT S473 phosphorylation without affecting the PDK1-mediated AKT T308 phosphorylation or TORC1 activity. The TORC2 activation was further supported by enhanced mTOR/RICTOR association and increased phosphorylation of additional TORC2 substrates, SGK1 and PKC?. The androgen-stimulated nuclear translocation of AR was associated with markedly-increased nuclear SIN1, a critical component of TORC2. Finally, the androgen-mediated TORC2/AKT activation targets a subset of AKT substrates including p27 and FOXO1, but not PRAS40. This study reveals a pathway linking AR to a selective activation of TORC2, the subsequent activation of AKT, and phosphorylation of a discrete set of AKT substrates that regulate cellular proliferation and survival. These findings establish that TORC2 can function as a central regulator of growth in response to signals that are distinct from those regulating TORC1, and support efforts to target TORC2 for cancer therapy.
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The altered expression of MiR-221/-222 and MiR-23b/-27b is associated with the development of human castration resistant prostate cancer.
Prostate
PUBLISHED: 09-15-2011
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We have previously identified seven miRs-miR-221, -222, -23b, -27b, -15a, -16-1, and -203, that are differentially expressed in the hormone sensitive LNCaP cell line and the hormone resistant LNCaP-abl cell line and hypothesized that these miRs may characterize certain subtypes of human castration resistant prostate cancer (CRPC).
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Intratumoral de novo steroid synthesis activates androgen receptor in castration-resistant prostate cancer and is upregulated by treatment with CYP17A1 inhibitors.
Cancer Res.
PUBLISHED: 08-25-2011
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Relapse of castration-resistant prostate cancer (CRPC) that occurs after androgen deprivation therapy of primary prostate cancer can be mediated by reactivation of the androgen receptor (AR). One important mechanism mediating this AR reactivation is intratumoral conversion of the weak adrenal androgens DHEA and androstenedione into the AR ligands testosterone and dihydrotestosterone. DHEA and androstenedione are synthesized by the adrenals through the sequential actions of the cytochrome P450 enzymes CYP11A1 and CYP17A1, so that CYP17A1 inhibitors such as abiraterone are effective therapies for CRPC. However, the significance of intratumoral CYP17A1 and de novo androgen synthesis from cholesterol in CRPC, and the mechanisms contributing to CYP17A1 inhibitor resistance/relapse, remain to be determined. We report that AR activity in castration-resistant VCaP tumor xenografts can be restored through CYP17A1-dependent de novo androgen synthesis, and that abiraterone treatment of these xenografts imposes selective pressure for increased intratumoral expression of CYP17A1, thereby generating a mechanism for development of resistance to CYP17A1 inhibitors. Supporting the clinical relevance of this mechanism, we found that intratumoral expression of CYP17A1 was markedly increased in tumor biopsies from CRPC patients after CYP17A1 inhibitor therapy. We further show that CRPC cells expressing a progesterone responsive T877A mutant AR are not CYP17A1 dependent, but that AR activity in these cells is still steroid dependent and mediated by upstream CYP11A1-dependent intraturmoral pregnenolone/progesterone synthesis. Together, our results indicate that CRPCs resistant to CYP17A1 inhibition may remain steroid dependent and therefore responsive to therapies that can further suppress de novo intratumoral steroid synthesis.
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SLCO2B1 and SLCO1B3 may determine time to progression for patients receiving androgen deprivation therapy for prostate cancer.
J. Clin. Oncol.
PUBLISHED: 05-23-2011
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Androgen deprivation therapy (ADT), an important treatment for advanced prostate cancer, is highly variable in its effectiveness. We hypothesized that genetic variants of androgen transporter genes, SLCO2B1 and SLCO1B3, may determine time to progression on ADT.
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Developing understanding of the roles of CD1d-restricted T cell subsets in cancer: reversing tumor-induced defects.
Clin. Immunol.
PUBLISHED: 04-12-2011
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Invariant natural killer T-cells (iNKT) are the best-known CD1d-restricted T-cells, with recently-defined roles in controlling adaptive immunity. CD1d-restricted T-cells can rapidly produce large amounts of Th1 and/or Th2//Treg/Th17-type cytokines, thereby regulating immunity. iNKT can stimulate potent anti-tumor immune responses via production of Th1 cytokines, direct cytotoxicity, and activation of effectors. However, Th2//Treg-type iNKT can inhibit anti-tumor activity. Furthermore, iNKT are decreased and/or reversibly functionally impaired in many advanced cancers. In some cases, CD1d-restricted T-cell cancer defects can be traced to CD1d(+) tumor interactions, since hematopoietic, prostate, and some other tumors can express CD1d. Ligand and IL-12 can reverse iNKT defects and therapeutic opportunities exist in correcting such defects alone and in combination. Early stage clinical trials have shown potential for reconstitution of iNKT IFN-gamma responses and evidence of activity in a subset of patients, with rational new approaches to capitalize on this progress ongoing, as will be discussed here.
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Androgen receptor gene expression in prostate cancer is directly suppressed by the androgen receptor through recruitment of lysine-specific demethylase 1.
Cancer Cell
PUBLISHED: 01-14-2011
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Androgen receptor (AR) is reactivated in castration-resistant prostate cancer (CRPC) through mechanisms including marked increases in AR gene expression. We identify an enhancer in the AR second intron contributing to increased AR expression at low androgen levels in CRPC. Moreover, at increased androgen levels, the AR binds this site and represses AR gene expression through recruitment of lysine-specific demethylase 1 (LSD1) and H3K4me1,2 demethylation. AR similarly represses expression of multiple genes mediating androgen synthesis, DNA synthesis, and proliferation while stimulating genes mediating lipid and protein biosynthesis. Androgen levels in CRPC appear adequate to stimulate AR activity on enhancer elements, but not suppressor elements, resulting in increased expression of AR and AR repressed genes that contribute to cellular proliferation.
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Intratumoral androgen biosynthesis in prostate cancer pathogenesis and response to therapy.
Endocr. Relat. Cancer
PUBLISHED: 01-01-2011
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The majority of prostate cancers (PCa) express high levels of androgen receptor (AR) and are dependent for their growth on testosterone produced by the testes, which is reduced in the prostate to the higher affinity ligand 5?-dihydrotestosterone (DHT). PCa growth can be suppressed by androgen deprivation therapy, which involves removal of testicular androgens (surgical or medical castration) or treatment with an AR antagonist (or a combination of both), but patients invariably relapse with tumors that have been termed castration recurrent/resistant PCa (CRPC). Importantly, AR transcriptional activity becomes reactivated at this CRPC stage of the disease and remains essential for tumor growth. The objective of this review is to outline one clinically important mechanism contributing to this AR reactivation, which is increased intratumoral synthesis of testosterone and DHT from weak androgens produced by the adrenal glands and possibly de novo from cholesterol. Early studies showed that a substantial fraction of CRPC patients responded to adrenalectomy or medical suppression of adrenal androgen synthesis using agents such as ketoconazole (CYP17A1 inhibitor), and a recent phase III study of a more potent and selective CYP17A1 inhibitor (abiraterone) has demonstrated an improvement in survival. With the pending FDA approval of abiraterone for CRPC, defining the molecular mechanisms contributing to CYP17A1 inhibitor resistance/relapse and AR reactivation is now critical to build on these advances.
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Isolation and functional use of human NKT cells.
Curr Protoc Immunol
PUBLISHED: 09-04-2010
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This unit details methods for the isolation, in vitro expansion, and functional characterization of human iNKT cells. The term iNKT derives from the fact that a large fraction of murine NKT cells recognize the MHC class I-like CD1d protein, are CD4+ or CD4-CD8- (double negative), and use an identical "invariant" TCRalpha chain, which is generated by precise Valpha14 and Jalpha281 (now renamed Jalpha18) rearrangements with either no N-region diversity or subsequent trimming to nearly identical amino-acid sequence (hence, iNKT). Basic Protocol 1 and Alternate Protocol 1 use multi-color FACS analysis to identify and quantitate rare iNKT cells from human samples. Basic Protocol 2 describes iNKT cell purification. Alternate Protocol 2 describes a method for high-speed FACS sorting of iNKT cells. Alternate Protocol 3 employs a cell sorting approach to isolate iNKT cell clones. A Support Protocol for secondary stimulation and rapid expansion of iNKT cells is also included. Basic Protocol 3 explains functional analysis of iNKT.
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Defective NKT cell activation by CD1d+ TRAMP prostate tumor cells is corrected by interleukin-12 with ?-galactosylceramide.
PLoS ONE
PUBLISHED: 03-16-2010
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Numerical and functional defects of invariant natural killer T cells (iNKT) have been documented in human and mouse cancers, resulting in a defect in IFN production in several malignancies. iNKT cells recognize glycolipids presented on CD1d molecules by dendritic and related cells, leading to their activation and thereby regulating immune reactions. Activated iNKT cells cytokine secretion and cytotoxicity can inhibit existing and spontaneous tumor growth, progression, and metastasis. We have identified functional iNKT cell defects in the murine TRAMP prostate cancer model. We found that iNKT cells show the ability to migrate into TRAMP prostate tumors. This infiltration was mediated through CCL2: CCR5 chemokine: receptor interaction. Prostate tumor cells expressing CD1d partially activated iNKT cells, as appreciated by up-regulation of CD25, PD-1 and the IL-12R. However, despite inducing up-regulation of these activation markers and, hence, delivering positive signals, prostate tumor cells inhibited the IL-12-induced STAT4 phosphorylation in a cell-cell contact dependent but CD1d-independent manner. Consequently, tumor cells did not induce secretion of IFNgamma by iNKT cells. Blocking the inhibitory Ly49 receptor on iNKT cells in the presence of alpha-GalCer restored their IFNgamma production in vivo and in vitro. However, Ly49 blockade alone was not sufficient. Importantly, this defect could be also be reversed into vigorous secretion of IFNgamma by the addition of both IL-12 and the exogenous CD1d ligand alpha-galactosylceramide, but not by IL-12 alone, both in vivo and in vitro. These data underscore the potential to optimize iNKT-based therapeutic approaches.
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Phosphoinositide 3-kinase pathway activation in phosphate and tensin homolog (PTEN)-deficient prostate cancer cells is independent of receptor tyrosine kinases and mediated by the p110beta and p110delta catalytic subunits.
J. Biol. Chem.
PUBLISHED: 03-15-2010
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Class IA phosphoinositide 3-kinase (PI3K) p110 catalytic subunits are activated upon Src homology 2 domain-mediated binding of their p85 regulatory subunits to tyrosine-phosphorylated pYXXM motifs in receptor tyrosine kinases (RTKs) or adaptor proteins. The PI3K pathway is activated by phosphate and tensin homolog (PTEN) loss in most prostate cancers (PCa), but the contribution of upstream RTKs that may be targeted therapeutically has not been assessed. Immunoblotting of p85-associated proteins in serum-starved PTEN-deficient LNCaP and C4-2 PCa cells showed a small set of discrete tyrosine-phosphorylated proteins, but these proteins were not recognized by an anti-pYXXM motif antibody and were not found in PTEN-deficient PC3 PCa cells. LC/MS/MS using label-free proteomics and immunoblotting showed that p85 was associated primarily with p110beta and p110delta. An interaction with ErbB3 was also detected but was independent of ErbB3 tyrosine phosphorylation and was not required for basal PI3K activity. Basal tyrosine phosphorylation of p110beta and p110delta could be blocked by c-Src inhibitors, but this did not suppress PI3K activity, which was similarly independent of Ras. Basal PI3K activity was mediated by p110beta in PC3 cells and by both p110beta and p110delta in LNCaP cells, whereas p110alpha was required for PI3K activation in response to RTK stimulation by heregulin-beta1. These findings show that basal PI3K activity in PTEN-deficient PCa cells is RTK-independent and can be mediated by p110beta and p110delta. Increased p110beta expression in PCa may be required for RTK-independent PI3K pathway activation in adult prostate epithelium with genetic or epigenetic PTEN down-regulation.
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Direct CD1d-mediated stimulation of APC IL-12 production and protective immune response to virus infection in vivo.
J. Immunol.
PUBLISHED: 11-30-2009
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CD1d-restricted NKT cells rapidly stimulate innate and adaptive immunity through production of Th1 and/or Th2 cytokines and induction of CD1d(+) APC maturation. However, therapeutic exploitation of NKT cells has been hampered by their paucity and defects in human disease. NKT cell-APC interactions can be modeled by direct stimulation of human APCs through CD1d in vitro. We have now found that direct ligation with multiple CD1d mAbs also stimulated bioactive IL-12 release from CD1d(+) but not CD1d knockout murine splenocytes in vitro. Moreover, all of the CD1d mAbs tested also induced IL-12 as well as both IFN-gamma and IFN-alpha in vivo from CD1d(+) but not CD1d-deficient recipients. Unlike IFN-gamma, CD1d-induced IFN-alpha was at least partially dependent on invariant NKT cells. Optimal resistance to infection with picornavirus encephalomyocarditis virus is known to require CD1d-dependent APC IL-12-induced IFN-gamma as well as IFN-alpha. CD1d ligation in vivo enhanced systemic IL-12, IFN-gamma, and IFN-alpha and was protective against infection by encephalomyocarditis virus, suggesting an alternative interpretation for previous results involving CD1d "blocking" in other systems. Such protective responses, including elevations in Th1 cytokines, were also seen with CD1d F(ab)(2)s in vivo, whereas an IgM mAb (with presumably minimal tissue penetration) was comparably effective at protection in vivo as well as cytokine induction both in vivo and in vitro. Although presumably acting immediately "downstream," CD1d mAbs were protective later during infection than the invariant NKT cell agonist alpha-galactosylceramide. These data indicate that NKT cells can be bypassed with CD1d-mediated induction of robust Th1 immunity, which may have therapeutic potential both directly and as an adjuvant.
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Phase II study of androgen synthesis inhibition with ketoconazole, hydrocortisone, and dutasteride in asymptomatic castration-resistant prostate cancer.
Clin. Cancer Res.
PUBLISHED: 11-03-2009
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Increasing evidence indicates that enhanced intratumoral androgen synthesis contributes to prostate cancer progression after androgen deprivation therapy. This phase II study was designed to assess responses to blocking multiple steps in androgen synthesis with inhibitors of CYP17A1 (ketoconazole) and type I and II 5alpha-reductases (dutasteride) in patients with castration-resistant prostate cancer (CRPC).
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Activity of dutasteride plus ketoconazole in castration-refractory prostate cancer after progression on ketoconazole alone.
Clin Genitourin Cancer
PUBLISHED: 10-10-2009
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Ketoconazole is a commonly used secondary hormonal therapy in castration-refractory prostate cancer (CRPC), but disease progression inevitably occurs. Both prostatic and metastatic lesions in patients with CRPC overexpress 5-alpha reductase (SRDA5) type I. We hypothesized that SRDA5 inhibition in combination with ketoconazole would mitigate progression after treatment with ketoconazole alone.
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Androgen receptor phosphorylation and activity are regulated by an association with protein phosphatase 1.
J. Biol. Chem.
PUBLISHED: 07-21-2009
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Androgen receptor (AR) is phosphorylated at multiple sites in response to ligand binding, but the functional consequences and mechanisms regulating AR phosphorylation remain to be established. We observed initially that okadaic acid, an inhibitor of the major PPP family serine/threonine phosphatases PP2A and protein phosphatase 1 (PP1), had cell type-dependent effects on AR expression. More specific inhibitors of PP2A (fostriecin) and PP1 (tautomycin and siRNA against the PP1alpha catalytic subunit) demonstrated that PP1 and protein phosphatase 2A had opposite effects on AR protein and transcriptional activity. PP1 inhibition enhanced proteasome-mediated AR degradation, while PP1alpha overexpression increased AR expression and markedly enhanced AR transcriptional activity. Coprecipitation experiments demonstrated an AR-PP1 interaction, while immunofluorescence and nuclear-cytoplasmic fractionation showed androgen-stimulated nuclear translocation of both AR and PP1 in prostate cancer cells. Studies with phosphospecific AR antibodies showed that PP1 inhibition dramatically increased phosphorylation of Ser-650, a site in the AR hinge region shown to mediate nuclear export. Significantly, PP1 inhibition caused a marked decrease in nuclear localization of the wild-type AR, but did not alter total or nuclear levels of a S650A mutant AR. These findings reveal a critical role of PP1 in regulating AR protein stability and nuclear localization through dephosphorylation of Ser-650. Moreover, AR may function as a PP1 regulatory subunit and mediate PP1 recruitment to chromatin, where it can modulate transcription and splicing.
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Reactivation of androgen receptor-regulated TMPRSS2:ERG gene expression in castration-resistant prostate cancer.
Cancer Res.
PUBLISHED: 07-07-2009
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It seems clear that androgen receptor (AR)-regulated expression of the TMPRSS2:ERG fusion gene plays an early role in prostate cancer (PC) development or progression, but the extent to which TMPRSS2:ERG is down-regulated in response to androgen deprivation therapy (ADT) and whether AR reactivates TMPRSS2:ERG expression in castration-resistant PC (CRPC) have not been determined. We show that ERG message levels in TMPRSS2:ERG fusion-positive CRPC are comparable with the levels in fusion gene-positive primary PC, consistent with the conclusion that the TMPRSS2:ERG expression is reactivated by AR in CRPC. To further assess whether TMPRSS2:ERG expression is initially down-regulated in response to ADT, we examined VCaP cells, which express the TMPRSS2:ERG fusion gene, and xenografts. ERG message and protein rapidly declined in response to removal of androgen in vitro and castration in vivo. Moreover, as observed in the clinical samples, ERG expression was fully restored in the VCaP xenografts that relapsed after castration, coincident with AR reactivation. AR reactivation in the relapsed xenografts was also associated with marked increases in mRNA encoding AR and androgen synthetic enzymes. These results show that expression of TMPRSS2:ERG, similarly to other AR-regulated genes, is restored in CRPC and may contribute to tumor progression.
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Androgen receptor expression in prostate cancer cells is suppressed by activation of epidermal growth factor receptor and ErbB2.
Cancer Res.
PUBLISHED: 06-02-2009
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Prostate cancers (PCa) that relapse after androgen deprivation therapies [castration-resistant PCa (CRPC)] express high levels of androgen receptor (AR) and androgen-regulated genes, and evidence from several groups indicates that ErbB family receptor tyrosine kinases [epidermal growth factor (EGF) receptor (EGFR) and ErbB2] may contribute to enhancing this AR activity. We found that activation of these kinases with EGF and heregulin-beta1 rapidly (within 8 hours) decreased expression of endogenous AR and androgen-regulated PSA in LNCaP PCa cells. AR expression was similarly decreased in LAPC4 and C4-2 cells, but not in the CWR22Rv1 PCa cell line. The rapid decrease in AR was not due to increased AR protein degradation and was not blocked by phosphatidylinositol 3-kinase (LY294002) or MEK (UO126) inhibitors. Significantly, AR mRNA levels in LNCaP cells were markedly decreased by EGF and heregulin-beta1, and experiments with actinomycin D to block new mRNA synthesis showed that AR mRNA degradation was increased. AR mRNA levels were still markedly decreased by EGF and heregulin-beta1 in LNCaP cells adapted to growth in androgen-depleted medium, although AR protein levels did not decline due to increased AR protein stability. These findings show that EGFR and ErbB2 can negatively regulate AR mRNA and may provide an approach to suppress AR expression in CRPC.
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Development of androgen receptor antagonists with promising activity in castration-resistant prostate cancer.
Cancer Cell
PUBLISHED: 05-30-2009
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Androgen receptor (AR) continues to play a central role in prostate cancers that relapse after androgen deprivation therapy, but these tumors are refractory to available AR antagonists. In a recent issue of Science, Tran et al. describe an antagonist that prevents AR recruitment to chromatin and shows efficacy in relapsed prostate cancer.
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Androgen receptor regulates a distinct transcription program in androgen-independent prostate cancer.
Cell
PUBLISHED: 04-22-2009
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The evolution of prostate cancer from an androgen-dependent state to one that is androgen-independent marks its lethal progression. The androgen receptor (AR) is essential in both, though its function in androgen-independent cancers is poorly understood. We have defined the direct AR-dependent target genes in both androgen-dependent and -independent cancer cells by generating AR-dependent gene expression profiles and AR cistromes. In contrast to what is found in androgen-dependent cells, AR selectively upregulates M-phase cell-cycle genes in androgen-independent cells, including UBE2C, a gene that inactivates the M-phase checkpoint. We find that epigenetic marks at the UBE2C enhancer, notably histone H3K4 methylation and FoxA1 transcription factor binding, are present in androgen-independent cells and direct AR-enhancer binding and UBE2C activation. Thus, the role of AR in androgen-independent cancer cells is not to direct the androgen-dependent gene expression program without androgen, but rather to execute a distinct program resulting in androgen-independent growth.
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The role of microRNA-221 and microRNA-222 in androgen-independent prostate cancer cell lines.
Cancer Res.
PUBLISHED: 04-07-2009
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Androgen-dependent prostate cancer typically progresses to castration-resistant prostate cancer (CRPC) after the androgen deprivation therapy. MicroRNAs (miR) are noncoding small RNAs (19-25nt) that play an important role in the regulation of gene expression. Recent studies have shown that miR expression patterns are significantly different in normal and neoplastic prostate epithelial cells. However, the importance of miRs in the development of CRPC has not yet been explored. By performing genome-wide expression profiling of miRs, we found that expression levels of several miRs, in particular miR-221 and miR-222, were significantly increased in CRPC cells (the LNCaP-derived cell line LNCaP-Abl), compared with those in the androgen-dependent prostate cancer cell line (LNCaP). Overexpression of miR-221 or miR-222 in LNCaP or another androgen-dependent cell line, LAPC-4, significantly reduced the level of the dihydrotestosterone (DHT) induced up-regulation of prostate-specific antigen (PSA) expression and increased androgen-independent growth of LNCaP cells. Knocking down the expression level of miR-221 and miR-222 with antagonist miRs in the LNCaP-Abl cell line restored the response to the DHT induction of PSA transcription and also increased the growth response of the LNCaP-Abl cells to the androgen treatment. Changing the expression level of p27/kip1, a known target of miR-221 and miR-222, alone in LNCaP cells affected the DHT-independent cell growth but did not significantly influence the response of PSA transcription to the DHT treatment. In conclusion, our data suggest the involvement of miR-221 and miR-222 in the development or maintenance of the CRPC phenotype.
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Mechanisms mediating androgen receptor reactivation after castration.
Urol. Oncol.
PUBLISHED: 03-21-2009
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Androgen deprivation is still the standard systemic therapy for metastatic prostate cancer (PCa), but patients invariably relapse with a more aggressive form of PCa termed hormone refractory, androgen independent, or castration resistant PCa (CRPC). Significantly, the androgen receptor (AR) is expressed at high levels in most cases of CRPC, and these tumors resume their expression of multiple AR-regulated genes, indicating that AR transcriptional activity becomes reactivated at this stage of the disease. The molecular basis for this AR reactivation remains unclear, but possible mechanisms include increased AR expression, AR mutations that enhance activation by weak androgens and AR antagonists, increased expression of transcriptional coactivator proteins, and activation of signal transduction pathways that can enhance AR responses to low levels of androgens. Recent data indicate that CRPC cells may also carry out intracellular synthesis of testosterone and DHT from weak adrenal androgens and may be able to synthesize androgens from cholesterol. These mechanisms that appear to contribute to AR reactivation after castration are further outlined in this review.
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Detection of TMPRSS2-ERG fusion gene expression in prostate cancer specimens by a novel assay using branched DNA.
Urology
PUBLISHED: 01-08-2009
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To develop a novel assay that uses branched DNA technology to measure TMPRSS2-ERG fusion, as genetic rearrangement of TMPRSS2 regulatory sequences and coding sequences of the ERG gene has been detected in nearly half of prostate cancers, but quantitative assays to detect such TMPRSS2-ERG gene fusion have been limited to real-time polymerase chain reaction (PCR) techniques that rely on reverse transcriptase-based amplification.
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EZH2 oncogenic activity in castration-resistant prostate cancer cells is Polycomb-independent.
Science
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Epigenetic regulators represent a promising new class of therapeutic targets for cancer. Enhancer of zeste homolog 2 (EZH2), a subunit of Polycomb repressive complex 2 (PRC2), silences gene expression via its histone methyltransferase activity. We found that the oncogenic function of EZH2 in cells of castration-resistant prostate cancer is independent of its role as a transcriptional repressor. Instead, it involves the ability of EZH2 to act as a coactivator for critical transcription factors including the androgen receptor. This functional switch is dependent on phosphorylation of EZH2 and requires an intact methyltransferase domain. Hence, targeting the non-PRC2 function of EZH2 may have therapeutic efficacy for treating metastatic, hormone-refractory prostate cancer.
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Clonal progression of prostate cancers from Gleason grade 3 to grade 4.
Cancer Res.
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Low-grade prostate cancers (Gleason pattern 3, G3) detected on needle biopsies are generally viewed as indolent and suitable for conservative management with only interval repeat biopsies to monitor by watchful waiting. Higher grade tumors eventually emerge in 20% to 30% of these cases, but this process may only reflect incomplete sampling on the initial biopsy, such that it remains unknown whether G3 tumors generally progress to higher grades. In this study, we examined a series of adjacent G3 and Gleason pattern 4 (G4) tumors in radical prostatectomy specimens and found that all were concordant for the TMPRSS2:ERG gene fusion. Using hybrid-capture and deep sequencing in four fusion-positive cases, we found that adjacent laser-capture microdissected G3 and G4 tumors had identical TMPRSS2:ERG fusion breakpoints, confirming their clonal origin. Two of these G3 tumors had deletion of a single PTEN gene that was also deleted in the adjacent G4, while the G4 tumors in two cases had additional PTEN losses. These findings establish that a subset of G3 tumors progress to G4 or emerge from a common precursor. Further, they show that G3 tumors that progress to G4 may have molecular features distinguishing them from G3 tumors that do not progress. Thus, determining the spectrum of these genetic or epigenetic features may allow for the identification of G3 tumors on needle biopsies that are truly indolent versus those that have the potential to progress or that may already be associated with a G4 tumor that was not sampled at the initial biopsy, therefore, requiring more aggressive surveillance or intervention.
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In silico discovery of androgen receptor antagonists with activity in castration resistant prostate cancer.
Mol. Endocrinol.
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Previously available androgen receptor (AR) antagonists (bicalutamide, flutamide, and nilutamide) have limited activity against AR in prostate cancers that relapse after castration [castration resistant prostate cancer (CRPC)]. However, recent AR competitive antagonists such as MDV3100, generated through chemical modifications to the current AR ligands, appear to have increased activity in CRPC and have novel mechanisms of action. Using pharmacophore models and a refined homology model of the antagonist-liganded AR ligand binding domain, we carried out in silico screens of small molecule libraries and report here on the identification of a series of structurally distinct nonsteroidal small molecule competitive AR antagonists. Despite their unique chemical architectures, compounds representing each of six chemotypes functioned in vitro as pure AR antagonists. Moreover, similarly to MDV3100 and in contrast to previous AR antagonists, these compounds all prevented AR binding to chromatin, consistent with each of the six chemotypes stabilizing a similar AR antagonist conformation. Additional studies with the lead chemotype (chemotype A) showed enhanced AR protein degradation, which was dependent on helix 12 in the AR ligand binding domain. Significantly, chemotype A compounds functioned as AR antagonists in vivo in normal male mice and suppressed AR activity and tumor cell proliferation in human CRPC xenografts. These data indicate that certain ligand-induced structural alterations in the AR ligand binding domain may both impair AR chromatin binding and enhance AR degradation and support continued efforts to develop AR antagonists with unique mechanisms of action and efficacy in CRPC.
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Adipose tissue invariant NKT cells protect against diet-induced obesity and metabolic disorder through regulatory cytokine production.
Immunity
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Invariant natural killer T (iNKT) cells are evolutionarily conserved innate T cells that influence inflammatory responses. We have shown that iNKT cells, previously thought to be rare in humans, were highly enriched in human and murine adipose tissue, and that as adipose tissue expanded in obesity, iNKT cells were depleted, correlating with proinflammatory macrophage infiltration. iNKT cell numbers were restored in mice and humans after weight loss. Mice lacking iNKT cells had enhanced weight gain, larger adipocytes, fatty livers, and insulin resistance on a high-fat diet. Adoptive transfer of iNKT cells into obese mice or in vivo activation of iNKT cells via their lipid ligand, alpha-galactocylceramide, decreased body fat, triglyceride levels, leptin, and fatty liver and improved insulin sensitivity through anti-inflammatory cytokine production by adipose-derived iNKT cells. This finding highlights the potential of iNKT cell-targeted therapies, previously proven to be safe in humans, in the management of obesity and its consequences.
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Doxycycline regulated induction of AKT in murine prostate drives proliferation independently of p27 cyclin dependent kinase inhibitor downregulation.
PLoS ONE
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The PI3 kinase/AKT pathway has been shown to increase degradation of the p27 cyclin dependent kinase inhibitor through phosphorylation of consensus AKT sites on p27 and SKP2, and AKT driven proliferation may be checked by feedback mechanisms that increase p27 expression and induce senescence. However, these AKT sites are not conserved in mouse, and it has not been clear whether AKT negatively regulates murine p27. Transgenic mice with a probasin promoter controlled prostate specific reverse tetracycline transactivator (ARR2Pb-rtTA) were generated and used to achieve doxycycline inducible expression of a tetracycline operon regulated constitutively active myristoylated AKT1 transgene (tetO-myrAKT). Doxycycline induction of myrAKT occurred within 1 day and rapidly induced proliferation (within 4 days) and the development of prostatic intraepithelial neoplasia (PIN) lesions in ventral prostate, which did not progress to prostate cancer. Cells in these lesions expressed high levels of p27, had increased proliferation, and there was apoptosis of centrally located cells. Doxycycline withdrawal resulted in apoptosis of cells throughout the lesions and rapid clearing of hyperplastic glands, confirming in vivo the critical antiapoptotic functions of AKT. Significantly, analyses of prostates immediately after initiating doxycycline treatment further showed that p27 expression was rapidly increased, coincident with the induction of myrAKT and prior to the development of hyperplasia and PIN. These findings establish in vivo that murine p27 is not negatively regulated by AKT and indicate that proliferation in PI3 kinase/AKT pathway driven mouse models is mediated by p27 independent mechanisms that may be distinct from those in human. Further studies using prostate specific doxycycline regulated transgene expression may be useful to assess the acute effects of inducing additional transgenes in adult murine prostate epithelium, and to assess the requirements for continued transgene expression in transgene induced tumors.
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Androgen receptor serine 81 phosphorylation mediates chromatin binding and transcriptional activation.
J. Biol. Chem.
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Our previous findings indicated that androgen receptor (AR) phosphorylation at serine 81 is stimulated by the mitotic cyclin-dependent kinase 1 (CDK1). In this report, we extended our previous study and confirmed that Ser-81 phosphorylation increases during mitosis, coincident with CDK1 activation. We further showed blocking cell cycle at G(1) or S phase did not disrupt androgen-induced Ser-81 phosphorylation and AR-dependent transcription, consistent with a recent report that AR was phosphorylated at Ser-81 and activated by the transcriptional CDK9. To assess the function of Ser-81 phosphorylation in prostate cancer (PCa) cells expressing endogenous AR, we developed a ligand switch strategy using a ligand-binding domain mutation (W741C) that renders AR responsive to the antagonist bicalutamide. An S81A/W741C double mutant AR stably expressed in PCa cells failed to transactivate the endogenous AR-regulated PSA or TMPRSS2 genes. ChIP showed that the S81A mutation prevented ligand-induced AR recruitment to these genes, and cellular fractionation revealed that the S81A mutation globally abrogated chromatin binding. Conversely, the AR fraction rapidly recruited to chromatin after androgen stimulation was highly enriched for Ser-81 phosphorylation. Finally, inhibition of CDK1 and CDK9 decreased AR Ser-81 phosphorylation, chromatin binding, and transcriptional activity. These findings indicate that Ser-81 phosphorylation by CDK9 stabilizes AR chromatin binding for transcription and suggest that CDK1-mediated Ser-81 phosphorylation during mitosis provides a pool of Ser-81 phosphorylation AR that can be readily recruited to chromatin for gene reactivation and may enhance AR activity in PCa.
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