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The androgen receptor (AR) is a transcriptional factor that has a pivotal role in the development of normal and also cancerous prostate. Therefore, analyzing AR signaling is essential to understand cancerogensis and proliferation of prostate cancer (PCa). Inhibitor of growth 1 (ING1) and ING2 are tumor suppressors with reduced expression in many cancer types. There are also indications of misregulation of ING1 and ING2 in PCa. However, the roles of ING1 and ING2 in PCa and AR signaling are poorly understood. Here, we show that surprisingly the ING1b knockdown (KD) represses AR-mediated transactivation on AR key target genes in the human LNCaP PCa cells. This is associated with growth reduction of LNCaP cells by ING1 KD. In line with this, using Ing1 knockout (KO) mice, we provide further evidence that ING1 deficiency downregulates prostate-specific AR target genes in vivo. Further analyses suggest that KD of ING1b results in induction of both cellular senescence and the cell cycle inhibitor p16 INK4a . The unexpected finding that the ING1 KD results in growth inhibition was further analyzed and can be explained by a compensatory mechanism through enhanced levels of ING2 protein in ING1-deficient condition. Accordingly, the data suggest that ING2 interacts with AR and hampers the AR transcriptional activation, causes growth arrest, and induces cellular senescence. The data further suggest that ING2 upregulates p16 INK4a , which is a novel target for ING2. Taken together, our data suggest that ING2 is a novel corepressor for AR. ING2 levels are increased upon downregulation of ING1 expression indicating a compensatory mechanism and suggests a novel crosstalk between ING1 and ING2 tumor suppressors to inhibit AR signaling and induce cellular senescence in PCa cells. • The tumor suppressors ING1 and 2 are dysregulated in human prostate cancer. • ING1 deficiency reduces AR-mediated gene expression in vitro and in vivo. • ING2, like ING1, inhibits AR-mediated transactivation and prostate cancer cell growth. • ING1 regulates ING2. • ING1 and ING2 crosstalk with each other to inhibit AR signaling in prostate cancer.

The development of prostate cancer (PCa) is regulated by the androgen-dependent activity of the androgen receptor (AR). Androgen-deprivation therapy (ADT) is therefore the gold standard treatment to suppress malignant progression of PCa. Nevertheless, due to the development of castration resistance, recurrence of disease after initial response to ADT is a major obstacle to successful treatment. As G-protein coupled receptors play a fundamental role in PCa physiology, they might represent promising alternative or combinatorial targets for advanced diseases. Here, we verified gene expression of the olfactory receptors (ORs) OR51E1 [prostate-specific G-protein coupled receptor 2 (PSGR2)] and OR51E2 (PSGR) in human PCa tissue by RNA-Seq analysis and RT-PCR and elucidated the subcellular localization of both receptor proteins in human prostate tissue. The OR51E1 agonist nonanoic acid (NA) leads to the phosphorylation of various protein kinases and growth suppression of the PCa cell line LNCaP. Furthermore, treatment with NA causes reduction of androgen-mediated AR target gene expression. Interestingly, NA induces cellular senescence, which coincides with reduced E2F1 mRNA levels. In contrast, treatment with the structurally related compound 1-nonanol or the OR2AG1 agonist amyl butyrate, neither of which activates OR51E1, did not lead to reduced cell growth or an induction of cellular senescence. However, decanoic acid, another OR51E1 agonist, also induces cellular senescence. Thus, our results suggest the involvement of OR51E1 in growth processes of PCa cells and its impact on AR-mediated signaling. These findings provide novel evidences to support the functional importance of ORs in PCa pathogenesis.

The COP9 signalosome (CSN) is a highly conserved protein complex, recently being crystallized for human. In mammals and plants the COP9 complex consists of nine subunits, CSN 1–8 and CSNAP. The CSN regulates the activity of culling ring E3 ubiquitin and plays central roles in pleiotropy, cell cycle, and defense of pathogens. Despite the interesting and essential functions, a thorough analysis of the CSN subunits in evolutionary comparative perspective is missing. Here we compared 61 eukaryotic genomes including plants, animals, and yeasts genomes and show that the most conserved subunits of eukaryotes among the nine subunits are CSN2 and CSN5. This may indicate a strong evolutionary selection for these two subunits. Despite the strong conservation of the protein sequence, the genomic structures of the intron/exon boundaries indicate no conservation at genomic level. This suggests that the gene structure is exposed to a much less selection compared with the protein sequence. We also show the conservation of important active domains, such as PCI (proteasome lid-CSN-initiation factor) and MPN (MPR1/PAD1 amino-terminal). We identified novel exons and alternative splicing variants for all CSN subunits. This indicates another level of complexity of the CSN. Notably, most COP9-subunits were identified in all multicellular and unicellular eukaryotic organisms analyzed, but not in prokaryotes or archaeas. Thus, genes encoding CSN subunits present in all analyzed eukaryotes indicate the invention of the signalosome at the root of eukaryotes. The identification of alternative splice variants indicates possible “mini-complexes” or COP9 complexes with independent subunits containing potentially novel and not yet identified functions.

The androgen receptor (AR) plays a leading role in the control of prostate cancer (PCa) growth. Interestingly, structurally different AR antagonists with distinct mechanisms of antagonism induce cell senescence, a mechanism that inhibits cell cycle progression, and thus seems to be a key cellular response for the treatment of PCa. Surprisingly, while physiological levels of androgens promote growth, supraphysiological androgen levels (SAL) inhibit PCa growth in an AR-dependent manner by inducing cell senescence in cancer cells. Thus, oppositional acting ligands, AR antagonists, and agonists are able to induce cellular senescence in PCa cells, as shown in cell culture model as well as ex vivo in patient tumor samples. This suggests a dual AR-signaling dependent on androgen levels that leads to the paradox of the rational to keep the AR constantly inactivated in order to treat PCa. These observations however opened the option to treat PCa patients with AR antagonists and/or with androgens at supraphysiological levels. The latter is currently used in clinical trials in so-called bipolar androgen therapy (BAT). Notably, cellular senescence is induced by AR antagonists or agonist in both androgen-dependent and castration-resistant PCa (CRPC). Pathway analysis suggests a crosstalk between AR and the non-receptor tyrosine kinase Src-Akt/PKB and the PI3K-mTOR-autophagy signaling in mediating AR-induced cellular senescence in PCa. In this review, we summarize the current knowledge of therapeutic induction and intracellular pathways of AR-mediated cellular senescence.

Prostate Cancer (PCa) is an important age-related disease being the most common cancer malignancy and the second leading cause of cancer mortality in men in Western countries. Initially, PCa progression is androgen receptor (AR)- and androgen-dependent. Eventually advanced PCa reaches the stage of Castration-Resistant Prostate Cancer (CRPC), but remains dependent on AR, which indicates the importance of AR activity also for CRPC. Here, we discuss various pathways that influence the AR activity in CRPC, which indicates an adaptation of the AR signaling in PCa to overcome the treatment of PCa. The adaptation pathways include interferences of the normal regulation of the AR protein level, the expression of AR variants, the crosstalk of the AR with cytokine tyrosine kinases, the Src-Akt-, the MAPK-signaling pathways and AR corepressors. Furthermore, we summarize the current treatment options with regard to the underlying molecular basis of the common adaptation processes of AR signaling that may arise after the treatment with AR antagonists, androgen deprivation therapy (ADT) as well as for CRPC, and point towards novel therapeutic strategies. The understanding of individualized adaptation processes in PCa will lead to individualized treatment options in the future.

Epidemiological and preclinical data have demonstrated the preventative effects of ω-3 polyunsaturated fatty acids, including docosahexaenoic acid (DHA), on prostate cancer. However, there are inconsistencies in these previous studies and the underlying mechanisms remain to be elucidated. In the present study, the androgen receptor (AR), which is a transcription factor involved in cell proliferation and prostate carcinogenesis, was identified as a target of DHA. It was revealed that DHA inhibited hormone‑dependent growth of LNCaP prostate cancer cells. Reverse transcription-quantitative polymerase chain reaction analysis revealed that treatment with DHA caused no alteration in the transcribed mRNA expression levels of the AR gene. However, immunoblotting revealed that this treatment reduces the protein expression level of the AR. The androgen‑induced genes were subsequently repressed by treatment with DHA. It was demonstrated that DHA exhibits no effect on the translation process of the AR, however, it promotes the proteasome‑mediated degradation of the AR. Therefore, the present study provided a novel mechanism by which DHA exhibits an inhibitory effect on growth of prostate cancer cells.

The prostate adenocarcinoma is the cancer with the highest incidence for men in Western countries. Targeting the androgen receptor (AR) by antagonists is used as hormone therapy for prostate cancer (PCa), however, eventually therapy resistance occurs in most patients. In most of these cancer the AR signaling is active and thus AR remains an important drug target. Since many years we are characterizing novel chemical structural platforms to provide a broader possibility for compounds that bind to and act as AR antagonists. Here, we describe the chemical synthesis of a battery of novel steroidal derivatives as nor-homo-, spiro-oxolan- and spiro-oxetan- steroids. They modulate the transcriptional activity of the human AR. As AR antagonists, the spiro-oxetan- steroid derivatives seem to be the most potent steroid derivatives. They inhibit the transcriptional activity of both wild-type AR as well as the AR mutant T877A. In line with this, these compounds bind to the human AR and inhibit the proliferation of the human androgen-dependent growing PCa cell line LNCaP. Interestingly, the castration-resistant AR expressing human PC3-AR cells are also growth inhibited. On mechanistic level, fluorescence resonance energy transfer (FRET) assays with living cells indicate that the androgen-induced N/C terminal interaction of the AR is inhibited by the investigated compounds. Using fluorescence recovery after photobleaching (FRAP) assays in living cells suggest a higher mobility of the AR in the cell nuclei in the presence of spiro-oxetan- steroidal antagonists. Together, these findings suggest that spiro-oxetan- steroids are very useful as a chemical platform for novel AR antagonists.

A 40 year old woman with a history of HIV, congestive heart failure secondary to an unknown congenital heart defect, and hypertension presented to our emergency department with worsening edema. On room air, oxygen saturation was 55 percent . On 5L of oxygen via nasal cannula, oxygen saturation was 88 percent . Physical examination was notable for central cyanosis, facial and lid edema, a II/VI holosystolic murmur across right chest radiating to entire right back hemithorax, decreased breath sounds at bases with pulmonary crackles, clubbing of fingers and edema of bilateral lower extremities. The patient related lifelong knowledge of a congenital heart defect, but had not been seen by a cardiologist as an adult. She was asymptomatic, yet sedentary until one year ago when she had the first of multiple hospitalizations for acute decompensated heart failure. A chest x-ray showed massive cardiomegaly with right-sided calcified aortic arch and patchy bilateral infiltrates. Transthoracic echocardiogram revealed severe right ventricular hypertrophy, ventricular septal defect, overriding aorta, and ejection fraction of 50 percent , consistent with unrepaired ToF. We utilized multimodality imaging techniques including CT angiography and Cardiac MRI which further defined her cardiac anatomy. Findings were consistent with unrepaired ToF with pulmonary atresia. The pulmonary arteries arose directly from the aorta in a confluent fashion via a large patent ductus arteriosus, major aortopulmonary collateral artery. The arterialized pulmonary arteries were aneurysmal with dissection and mural thrombus formation.This case illustrates how a patient with a rare presentation of unrepaired ToF with pulmonary atresia can go undiagnosed into adulthood. Early recognition utilizing a multimodality imaging approach can lead to proper diagnosing and hopeful surgical repair which can provide considerable improvement in functional status and long-term survival.

Background/aims: Although polycystic ovary syndrome (PCOS) is a common endocrinopathy the pathogenesis is not entirely understood. Typically, high androgen levels are associated with increased virilization. We report 2 rare groups of patients with either unexpectedly high testosterone levels despite low virilization as well as patients with low testosterone levels despite high grade of virilization. One possibility for the atypical PCOS may be based on an altered androgen receptor (AR) signaling. Methods: 6 patients and when available the parents were included in this study. Alterations of the metaphase chromosomes by GTG staining, the length of both the trinucleotide CAG- and GGC-repeats of the androgen receptor (AR) gene was determined by PCR, further the entire AR gene was sequenced and analyzed. Results: The GTG banding revealed no chromosomal alterations and the range of CAG- and GGC-repeat lengths are within the normal range. Interestingly, by sequencing of the entire AR gene few genetic mutations were identified. Conclusion: The detected mutations do not alter the AR protein sequence but they change the codon usage towards less frequent codons that potentially may alter AR protein levels and androgen signaling. In addition to this, we postulate also other causes for manifestation of atypical PCOS, which may include AR-coregulators or epigenetic alterations. To our knowledge this is the first report of combining chromosomal analysis of PCOS patients with full sequencing of the human AR gene and linking codon usage to PCOS.

This article presents the kaonic atom studies performed at the INFN National Laboratory of Frascati (Laboratori Nazionali di Frascati dell&rsquo, INFN, LNF-INFN) since the opening of this field of research at the DA &Phi, NE collider in early 2000. Significant achievements have been obtained by the DA &Phi, NE Exotic Atom Research (DEAR) and Silicon Drift Detector for Hadronic Atom Research by Timing Applications (SIDDHARTA) experiments on kaonic hydrogen, which have required the development of novel X-ray detectors. The 2019 installation of the new SIDDHARTA-2 experiment to measure kaonic deuterium for the first time has been made possible by further technological advances in X-ray detection.

Heat shock proteins (HSPs) and chaperones are highly conserved stress-induced factors. They regulate not only protein folding and stability but are also actively involved in protein transport and transcriptional regulation. HSPs have cytoprotective roles and are essential for cancer cell survival. Noteworthy, HSPs are often upregulated in cancer. Therefore, HSPs emerged as drug targets for cancer therapy. Especially for prostate cancer (PCa) therapy, a battery of different compounds has been identified that act with different modes to inhibit PCa growth. The androgen receptor (AR) is a major player in PCa progression and is a well-known interacting factor of HSPs. Since the AR function is very dependent on HSP activity, many emerging compounds address the AR-associated HSPs as novel drug targets. Here, we provide an insight into the different classes of HSPs, their association with the human AR, the role of HSPs in human PCa development and review also the targeting of HSPs in human PCa. Further, the function and the underlying molecular mechanisms of specific compounds that are currently under investigation for the use against PCa growth will be comprehensively summarized.

Androgen receptor (AR) antagonists are important compounds for the treatment of prostate cancer (PCa). The atraric acid (AA), a natural compound, binds to the AR and acts as a specific AR antagonist. Interestingly, AA represents a novel chemical platform that could serve as a potential basis for new AR antagonists. Therefore, one objective of this study was to analyze the chemical/structural requirements for AR antagonism and to obtain predictions of where and how AA binds to the AR. Further, this study describes the chemical synthesis of 12 AA derivatives and their analysis using a combination of computational and functional assays. Functional analysis of AA derivatives indicated that none activated the AR. Both the para-hydroxyl group and the benzene ortho- and the meta-methyl groups of AA appeared to be essential to antagonize androgen-activated AR activity. Furthermore, extension of the hydrophobic side chain of AA led to slightly stronger AR antagonism. In silico data suggest that modifications to the basic AA structure change the hydrogen-bonding network with the AR ligand binding domain (LBD), so that the para-hydroxyl group of AA forms a hydrogen bond with the LBD, confirming the functional importance of this group for AR antagonism. Moreover, in silico modeling also suggested that the ortho- and meta- methyl groups of AA interact with hydrophobic residues of the ligand pocket of AR, which might explain their functional importance for antagonism. Thus, these studies identify the chemical groups of AA that play key roles in allowing the AA-based chemical platform to act as an AR antagonist.

The RNA-seq data generated during this work has been submitted to Gene Expression Omnibus and is available for viewing at the following link http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token = ytazouoixxedjal&acc=GSE63700. Background: The androgen receptor (AR) is a major drug target in prostate cancer (PCa). We profiled the AR-regulated kinome to identify clinically relevant and druggable effectors of AR signaling. Methods: Using genome-wide approaches, we interrogated all AR regulated kinases. Among these, choline kinase alpha (CHKA) expression was evaluated in benign (n = 195), prostatic intraepithelial neoplasia (PIN) (n = 153) and prostate cancer (PCa) lesions (n = 359). We interrogated how CHKA regulates AR signaling using biochemical assays and investigated androgen regulation of CHKA expression in men with PCa, both untreated (n = 20) and treated with an androgen biosynthesis inhibitor degarelix (n = 27). We studied the effect of CHKA inhibition on the PCa transcriptome using RNA sequencing and tested the effect of CHKA inhibition on cell growth, clonogenic survival and invasion. Tumor xenografts (n = 6 per group) were generated in mice using genetically engineered prostate cancer cells with inducible CHKA knockdown. Data were analyzed with χ 2 tests, Cox regression analysis, and Kaplan-Meier methods. All statistical tests were two-sided. Results: CHKA expression was shown to be androgen regulated in cell lines, xenografts, and human tissue (log fold change from 6.75 to 6.59, P = .002) and was positively associated with tumor stage. CHKA binds directly to the ligand-binding domain (LBD) of AR, enhancing its stability. As such, CHKA is the first kinase identified as an AR chaperone. Inhibition of CHKA repressed the AR transcriptional program including pathways enriched for regulation of protein folding, decreased AR protein levels, and inhibited the growth of PCa cell lines, human PCa explants, and tumor xenografts. Conclusions:CHKA can act as an AR chaperone, providing, to our knowledge, the first evidence for kinases as molecular chaperones, making CHKA both a marker of tumor progression and a potential therapeutic target for PCa. Publisher PDF

The epigenetic regulatory tumor suppressor, INhibitor of Growth 1 (ING1), obtained more focus since it has been suggested as one of the aging-related candidate genes among healthy elderly individuals. ING1 belongs to the ING family proteins characterized by a plant homeodomain (PHD), which is important for recognizing and binding to histone marks, thus allowing ING to regulate genes expression through histone modification and chromatin changes. Interestingly, the PHD of ING proteins is highly conserved among species between mammals, insects and plants. The ING factors regulate the program of cellular senescence and DNA repair, which are suggested to have a protective role in inhibiting cancer cells proliferation. Here, we provide an insight into the functional role of ING factors in development and tumor cells.

We describe a rare case of atypical pyoderma gangrenosum of the dorsal hand in a patient who presented with the histopathologic diagnosis of squamous cell carcinoma (SCC). Atypical pyoderma gangrenosum is a rare variant of pyoderma gangrenosum that occurs on the upper extremities, presenting as a cutaneous ulcer. The correct treatment is nonsurgical, and surgical intervention often results in trauma-induced expansion of lesions. Pyoderma gangrenosum, most commonly mistaken for infection, has resulted in amputation. Histologic and clinical presentation 1 month after onset of symptoms in the patient reported here mimicked SCC. Awareness by hand surgeons of the possible histologic misdiagnosis of SCC at an early stage in the disease evolution and a high index of suspicion in the face of biopsy-proven SCC may prevent unnecessary digit amputation.

Electron beam radiation is a commonly used and effective treatment modality for cutaneous malignancies. Secondary cutaneous malignancies arising in electron beam treatment sites are rarely reported. We report on seven patients who developed multiple secondary tumours arising within and immediately around electron beam radiation treatment sites in the setting of treatment for diffuse primary disease or as adjunctive therapy for aggressive cutaneous malignancies.

BACKGROUND: The androgen receptor (AR) is a major drug target in prostate cancer (PCa). We profiled the AR-regulated kinome to identify clinically relevant and druggable effectors of AR signaling.METHODS: Using genome-wide approaches, we interrogated all AR regulated kinases. Among these, choline kinase alpha (CHKA) expression was evaluated in benign (n = 195), prostatic intraepithelial neoplasia (PIN) (n = 153) and prostate cancer (PCa) lesions (n = 359). We interrogated how CHKA regulates AR signaling using biochemical assays and investigated androgen regulation of CHKA expression in men with PCa, both untreated (n = 20) and treated with an androgen biosynthesis inhibitor degarelix (n = 27). We studied the effect of CHKA inhibition on the PCa transcriptome using RNA sequencing and tested the effect of CHKA inhibition on cell growth, clonogenic survival and invasion. Tumor xenografts (n = 6 per group) were generated in mice using genetically engineered prostate cancer cells with inducible CHKA knockdown. Data were analyzed with χ(2) tests, Cox regression analysis, and Kaplan-Meier methods. All statistical tests were two-sided.RESULTS: CHKA expression was shown to be androgen regulated in cell lines, xenografts, and human tissue (log fold change from 6.75 to 6.59, P = .002) and was positively associated with tumor stage. CHKA binds directly to the ligand-binding domain (LBD) of AR, enhancing its stability. As such, CHKA is the first kinase identified as an AR chaperone. Inhibition of CHKA repressed the AR transcriptional program including pathways enriched for regulation of protein folding, decreased AR protein levels, and inhibited the growth of PCa cell lines, human PCa explants, and tumor xenografts.CONCLUSIONS: CHKA can act as an AR chaperone, providing, to our knowledge, the first evidence for kinases as molecular chaperones, making CHKA both a marker of tumor progression and a potential therapeutic target for PCa.

A plant extract from the fruits of saw palmetto, which is currently used to treat the androgen-dependent benign prostatic hyperplasia and PCa, served as source for new structure variants. We investigated the antiandrogenic potential of an ethanolic total extract and one of its main aromatic components anthranilic acid. An androgen receptor-antagonistic (antiandrogenic) effect of the extract was evident, and although anthranilic acid itself revealed no remarkable effect, its methyl ester, methyl anthranilate, exhibited antiandrogenic potential. Based on this chemical structure, we synthesized and investigated the antiandrogenic activity of four AnA ester derivatives, which were either novel or only little described in literature. These AnA esters inhibit the androgen-dependent transactivation of both the wild-type (wt) androgen receptor and the androgen receptor point mutant T877A, which often occurs in refractory PCa. Moreover, they inhibit the androgen receptor-induced expression of the endogenous prostate-specific antigen. Importantly, AnA esters repress the growth of human PCa cells. Deletion analyses of androgen receptor propose that the antiandrogenic effect of anthranilic acid esters is mediated by the ligand-binding domain, most likely through direct binding, without affecting androgen receptor protein levels. Taken together, the data suggest antiandrogenic potential of anthranilic acid ester derivatives, which can serve as lead structures for novel antiandrogens.

The interaction of antikaons with nucleons and nuclei in the low-energy regime represents an active research field in hadron physics with still many important open questions. The investigation of light kaonic atoms is, in this context, a unique tool to obtain precise information on this interaction. The energy shift and broadening of the lowest-lying states of such atoms, induced by the kaon-nucleus strong interaction, can be determined with high precision from atomic X-ray spectroscopy. This experimental method provides unique information to understand the low energy kaon-nucleus interaction at threshold. The lightest atomic systems, kaonic hydrogen and kaonic deuterium, deliver the isospin-dependent kaon-nucleon scattering lengths. The most precise kaonic hydrogen measurement to date, together with an exploratory measurement of kaonic deuterium, were carried out by the SIDDHARTA collaboration at the DAΦNE electron-positron collider of LNF-INFN, by combining the excellent quality kaon beam delivered by the collider with new experimental techniques, as fast and precise X-ray detectors: Silicon Drift Detectors. The measurement of kaonic deuterium will be realized in the near future by SIDDHARTA-2, a major upgrade of SIDDHARTA. In this paper an overview of the main results obtained by SIDDHARTA together with the future plans, are given.

Prostate cancer (PCa) ranks the most frequently diagnosed male cancer in the Western world (1). It develops first as an androgen-dependent tumor in which androgens promote the growth of the PCa cells. Eventually, it develops into a castration resistant stage that no longer depends on androgens.

The COP9 complex (signalosome) is a known regulator of the proteasome/ubiquitin pathway. Furthermore it regulates the activity of the cullin-RING ligase (CRL) families of ubiquitin E3-complexes. Besides the CRL family, the anaphase-promoting complex (APC/C) is a major regulator of the cell cycle. To investigate a possible connection between both complexes we assessed interacting partners of COP9 using an in vivo protein-protein interaction assay. Hereby, we were able to show for the first time that CSN2, a subunit of the COP9 signalosome, interacts physically with APC/C. Furthermore, we detected a functional influence of the COP9 complex regarding the stability of several targets of the APC/C. Consistent with these data we showed a genetic instability of cells overexpressing CSN2.

We have identified and profiled a set of androgen receptor (AR) binding compounds representing two nonsteroidal scaffolds from a public chemical database supplied by Asinex with virtual screening procedure incorporating our recently published 3D QSAR model of AR ligands. The diphenyl- and phenylpyridine-based compounds act as antagonists in wild-type AR in CV1 cells and also retain this antagonistic character in CV1 cells expressing T877A mutant receptor. This mutation is frequently associated with prostate cancer. Two of the compounds repress the androgen-dependent cell growth of LNCaP prostate cancer cells expressing the T877A AR mutant. Molecular modeling of the observed in vitro antagonism with induced fit docking suggests that W741 and M895 could be mechanistically involved in the initiation of the antagonism. The results indicate finding of nonsteroidal AR antagonist compounds from a public chemical database with computational methods. Compounds could serve as a novel platform to develop more potent AR antagonists with inhibitory activity in both wild-type and T877A mutant AR.

Prostate cancer is one of the most prominent malignancies of elderly men in many Western countries including Europe and the United States with increasing trend worldwide. The growth of normal prostate as well as of prostate carcinoma cells depends on functional androgen receptor (AR) signaling. AR manifests the biological actions of androgens and its transcriptional activity is known to be influenced by signal transduction pathways. Here we show that Src, a nonreceptor tyrosine kinase, is overexpressed in androgen-independent prostate carcinoma C4-2 cells. Interestingly, the expression of Src was found to progressively increase (up to threefold) in transgenic adenocarcinoma of mouse prostate mice as a function of age and cancer progression. Blocking Src kinase function by a specific inhibitor, PP2, resulted in decreased AR transactivation function on two different reporters, mouse mammary tumor virus (MMTV) and prostate-specific antigen (PSA). Consistent with this, overexpression of a functional Src mutant also led to a dramatic decrease in AR transactivation potential in a hormone-dependent manner. Interference with Src function in C4-2 cells led to decreased recruitment of AR on the target gene PSA enhancer and also resulted in the abrogation of hormone-dependent PSA transcript induction. Src inhibition also led to a dramatic decrease in the cell invasion in addition to decreasing the cellular growth. We suggest that targeting Src kinase could be an effective strategy to inhibit prostate cancer growth and metastasis.

The tumor suppressor p33ING1 is involved in DNA repair and cell cycle regulation. Furthermore, p33ING1 is a transcriptional silencer that recognizes the histone mark for trimethylated lysine 4 at histone H3. Interestingly, expression of p33ING1 and p33ING2 is able to induce premature senescence in primary human fibroblasts. The corepressor Alien is involved in gene silencing mediated by selected members of nuclear hormone receptors. In addition, Alien acts as a corepressor for E2F1, a member of the E2F cell cycle regulatory family. Furthermore, recent findings suggest that Alien is complexed with transcription factors participating in DNA repair and chromatin. Here, using a proteomic approach by surface-enhanced laser desorption ionization and mass spectrometry (SELDI-MS) combined with immunological techniques, we show that Alien interacts in vivo with the tumor suppressor p33ING1 as well as with the related tumor suppressor candidate p33ING2. The interaction of Alien with p33ING1 and p33ING2 was confirmed in vitro with GST-pull-down, suggesting a direct binding of Alien to these factors. The binding domain was mapped to a central region of Alien. Functionally, the expression of p33ING1 or p33ING2 enhances the Alien-mediated silencing, suggesting that the interaction plays a role in transcriptional regulation. Thus, the findings suggest that the identified interaction between Alien and the tumor suppressors p33ING1 and p33ING2 reveals a novel cellular protein network.

The assembly of nucleosomes into chromatin is essential for the compaction of DNA and inactivation of the DNA template to modulate and repress gene expression. The nucleosome assembly protein 1, NAP1, assembles nucleosomes independent of DNA synthesis and was shown to enhance coactivator-mediated gene expression, suggesting a role for NAP1 in transcriptional regulation. Here, we show that Alien, known to harbor characteristics of a corepressor of nuclear hormone receptors such as of the vitamin D receptor (VDR), binds in vivo and in vitro to NAP1 and modulates its activity by enhancing NAP1-mediated nucleosome assembly on DNA. Furthermore, Alien reduces the accessibility of the histones H3 and H4 for NAP1-promoted assembly reaction. This indicates that Alien sustains and reinforces the formation of nucleosomes. Employing deletion mutants of Alien suggests that different regions of Alien are involved in enhancement of NAP1-mediated nucleosome assembly and in inhibiting the accessibility of the histones H3 and H4. In addition, we provide evidence that Alien is associated with chromatin and with micrococcus nuclease-prepared nucleosome fractions and interacts with the histones H3 and H4. Furthermore, chromatin immunoprecipitation and reimmunoprecipitation experiments suggest that NAP1 and Alien localize to the endogenous CYP24 promoter in vivo, a VDR target gene. Based on these findings, we present here a novel pathway linking corepressor function with nucleosome assembly activity.

Prostate cancer cell growth is initially androgen dependent. Androgen antagonists are used in prostate cancer therapy to inactivate the transcriptional activity of the human androgen receptor (hAR) and to inhibit the proliferation of prostate cancer. Here, we have characterized Alien with characteristics of a corepressor as a novel interacting factor for the antagonist bound hAR. Alien is recruited to hAR in the presence of the AR antagonist cyproterone acetate (CPA). The interaction of Alien with hAR is verified in vivo and in vitro by a modified mammalian two-hybrid system, coimmunoprecipitation, chromatin immunoprecipitation, and in vitro binding assays. In contrast to other nuclear receptors, Alien binds to the amino-terminus of hAR with the receptor SUMOylation (small ubiquitin modifier) sites being involved. Furthermore, cellular localization of Alien is changed towards a predominant nuclear localization upon treatment of prostate cancer cells with CPA. Notably, stable expression of Alien in LNCaP cells inhibits both endogenous prostate-specific antigen expression and proliferation of these cells in the presence of CPA but not in the presence of an AR agonist. These findings underline the importance of corepressors for inhibition of prostate cancer cell growth by androgen antagonists.

Interacting proteins are often involved in the same cellular processes, and thus the identification of interacting partners of a given protein with unknown function may give insight into the physiological role of this protein. For the detection of protein-protein interactions of the corepressor Alien we used a proteomic approach comprising mass spectrometry and immunological techniques. We assessed solely endogenously expressed proteins. In this study we present for the first time that Alien is interacting within a network of proteins involved in transcriptional regulation, DNA repair, and cell cycle in vivo. In this way we detected protein interactions of Alien involving nucleophosmin, ERCC3, TRIP11, as well as CRSP3.

Spinal-bulbar muscle atrophy (SBMA) or also named Kennedy's Disease is caused by a polyglutamine expansion (PolyQ) of the coding region of the androgen receptor (AR). The AR is a ligand-controlled transcription factor and member of the nuclear hormone receptor superfamily. The central characteristics of the SBMA pathogenicity are muscle weakness, the loss of motoneurons and the occurrence of AR-containing protein aggregates that are observed in spinal cord motoneurons and skeletal muscles induced by the AR-PolyQ expansion in the presence of androgens. The PolyQ triggers a misfolding in the AR-PolyQ and leads to protein aggregation in spinal cord motoneurons and muscle cells. The AR-PolyQ toxicity is activated by the AR ligand testosterone and dihydrotestosterone that activate the receptor and triggers nuclear toxicity by inducing AR nuclear translocation. In line with this, androgen treatment of SBMA patients worsened the SBMA symptoms. SBMA has been modeled in AR-overexpressing and AR-PolyQ-knock-in animals, but precisely how the PolyQ expansion leads to neurodegeneration is unclear. The androgen-induced toxicity and androgen-dependent nuclear accumulation of AR-PolyQ protein seems to be central to the pathogenesis. Therefore, the inhibition of the androgen-activated AR-PolyQ might be a therapeutic option. Here the use of AR antagonists for treatment option of SBMA will be reviewed and discussed.