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Nucleosome repositioning in cancer highlights changes of many aspects of genome organisation and regulation. Understanding it is important both from the fundamental point of view and for medical diagnostics based on cell-free DNA (cfDNA), which originates from genomic DNA regions protected from digestion by nucleosomes. Here we generated ultra-high resolution nucleosome maps in paired tumour and normal tissues from the same breast cancer patients using MNase-assisted histone H3 ChIP-seq and compared them with the corresponding cfDNA from blood plasma. We detected cancer-specific single-nucleosome repositioning at key regulatory regions in a patient-specific manner, as well as common patterns across patients. The nucleosomes gained in tumour versus normal tissue were particularly informative of cancer pathways, with 20-fold enrichment at CpG islands, a large fraction of which marked promoters of genes encoding DNA-binding proteins. Tumour tissues were characterised by 5-10 bp decrease in average distances between nucleosomes (nucleosome repeat length, NRL). These effects were modulated by GC content and DNA sequence repeats and correlated with differential DNA methylation and binding of linker histone variants H1.4 and H1X. Our findings provide missing mechanistic understanding of nucleosome positioning in tumour tissues and can be valuable for nucleosomics analyses in liquid biopsies.

Supplementary Figures 1-6, Table 1 from FUS/TLS Is a Novel Mediator of Androgen-Dependent Cell-Cycle Progression and Prostate Cancer Growth

DNAzymes are short pieces of DNA with catalytic activity, capable of cleaving RNA. DNAzymes have multiple applications as biosensors and in therapeutics. The high specificity and low toxicity of these molecules make them particularly suitable as therapeutics, and clinical trials have shown that they are effective in patients. However, the development of DNAzymes has been limited due to the lack of specific tools to identify efficient molecules, and users often resort to time-consuming/costly large-scale screens. Here, we propose a computational methodology to identify 10–23 DNAzymes that can be used to triage thousands of potential molecules, specific to a target RNA, to identify those that are predicted to be efficient. The method is based on a logistic regression and can be trained to incorporate additional DNAzyme efficiency data, improving its performance with time. We first trained the method with published data, and then we validated, and further refined it, by testing additional newly synthesized DNAzymes in the laboratory. We found that although binding free energy between the DNAzyme and its RNA target is the primary determinant of efficiency, other factors such as internal structure of the DNAzyme also have an important effect. A program implementing the proposed method is publicly available.

COVID-19 symptoms and mortality are largely due to its devastating effects in the lungs. The disease is caused by the SARS (Severe Acute Respiratory Syndrome)-CoV-2 coronavirus, which requires host cell proteins such as ACE2 (angiotensin-converting enzyme 2) and TMPRSS2 (transmembrane serine protease 2) for infection of lung epithelia. The expression and function of the steroid hormone receptor family is important in many aspects that impact on COVID-19 effects in the lung – notably lung development and function, the immune system, and expression of TMPRSS2 and ACE2. This review provides a brief summary of current knowledge on the roles of the steroid hormone receptors [androgen receptor (AR), glucocorticoid receptor (GR), progesterone receptor (PR), mineralocorticoid receptor (MR) and oestrogen receptor (ER)] in the lung, their effects on host cell proteins that facilitate SARS-CoV-2 uptake, and provides a snapshot of current clinical trials investigating the use of steroid receptor (SR) ligands to treat COVID-19.

Mesenchymal stem cells (MSCs) belong to the tumour microenvironment and have been implicated in tumour progression. We found that the number of MSCs significantly increased in tumour-burdened mice driven by Fas-threshold signalling. Consequently, MSCs lacking Fas lost their ability to induce metastasis development in a pancreatic cancer model. Mixing of MSCs with pancreatic cancer cells led to sustained production of the pro-metastatic cytokines CCL2 and IL6 by the stem cells. The levels of these cytokines were dependent on the number of MSCs, linking Fas-mediated MSC-proliferation to their capacity to promote tumour progression. Furthermore, we discovered that CCL2 and IL6 were induced by pancreatic cancer cell-derived IL1. Importantly, analysis of patient transcriptomic data revealed that high FasL expression correlates with high levels of MSC markers as well as increased IL6 and CCL2 levels in pancreatic tumours. Moreover, both FasL and CCL2 are linked to elevated levels of markers specific for monocytes known to possess further pro-metastatic activities. These results confirm our experimental findings of a FasL-MSC-IL1-CCL2/IL6 axis in pancreatic cancer and highlights the role of MSCs in tumour progression.

SARS-CoV-2 attacks various organs, most destructively the lung, and cellular entry requires two host cell surface proteins: ACE2 and TMPRSS2. Downregulation of one or both of these is thus a potential therapeutic approach for COVID-19. TMPRSS2 is a known target of the androgen receptor, a ligand-activated transcription factor; androgen receptor activation increases TMPRSS2 levels in various tissues, most notably prostate. We show here that treatment with the antiandrogen enzalutamide—a well-tolerated drug widely used in advanced prostate cancer—reduces TMPRSS2 levels in human lung cells and in mouse lung. Importantly, antiandrogens significantly reduced SARS-CoV-2 entry and infection in lung cells. In support of this experimental data, analysis of existing datasets shows striking co-expression of AR and TMPRSS2, including in specific lung cell types targeted by SARS-CoV-2. Together, the data presented provides strong evidence to support clinical trials to assess the efficacy of antiandrogens as a treatment option for COVID-19., TMPRSS2 is regulated by androgen receptor signalling in the prostate, however it is unclear if blocking this signalling is beneficial in the context of SARS-CoV-2 lung infection. Here the authors show that antiandrogen treatment downregulates TMPRSS2 in the lung and reduces viral entry and infection.

Prostate cancer (PCa) is the second most commonly occurring cancer in men, with over a million new cases every year worldwide. Tumor growth and disease progression is mainly dependent on the Androgen Receptor (AR), a ligand dependent transcription factor. Standard PCa therapeutic treatments include androgen-deprivation therapy and AR signaling inhibitors. Despite being successful in controlling the disease in the majority of men, the high frequency of disease progression to aggressive and therapy resistant stages (termed castrate resistant prostate cancer) has led to the search for new therapeutic targets. The p90 ribosomal S6 kinase (RSK1-4) family is a group of highly conserved Ser/Thr kinases that holds promise as a novel target. RSKs are effector kinases that lay downstream of the Ras/Raf/MEK/ERK signaling pathway, and aberrant activation or expression of RSKs has been reported in several malignancies, including PCa. Despite their structural similarities, RSK isoforms have been shown to perform nonredundant functions and target a wide range of substrates involved in regulation of transcription and translation. In this article we review the roles of the RSKs in proliferation and motility, cell cycle control and therapy resistance in PCa, highlighting the possible interplay between RSKs and AR in mediating disease progression. In addition, we summarize the current advances in RSK inhibitor development and discuss their potential clinical benefits.

The COVID-19 pandemic, caused by the novel human coronavirus SARS-CoV-2 coronavirus, attacks various organs but most destructively the lung. It has been shown that SARS-CoV-2 entry into lung cells requires two host cell surface proteins: ACE2 and TMPRSS2. Downregulation of one or both of these is thus a potential therapeutic approach for COVID-19. TMPRSS2 is a known target of the androgen receptor, a ligand-activated transcription factor; activation of the androgen receptor increases TMPRSS2 levels in various tissues, most notably the prostate. We show here that treatment with the antiandrogen enzalutamide – a well-tolerated drug widely used in advanced prostate cancer – reduces TMPRSS2 levels in human lung cells. Further, enzalutamide treatment of mice dramatically decreased Tmprss2 levels in the lung. To determine therapeutic potential, we assessed uptake of SARS-CoV-2 Spike protein pseudotyped lentivirus and live SARS-CoV-2 into human lung cells and saw a significant reduction in viral entry and infection upon treatment with the antiandrogens enzalutamide and bicalutamide. In support of this new experimental data, analysis of existing datasets shows striking co-expression of AR and TMPRSS2, including in specific lung cell types that are targeted by SARS-CoV-2. Together, the data presented provides strong evidence to support clinical trials to assess the efficacy of antiandrogens as a treatment option for COVID-19.

SARS-CoV-2 is the novel coronavirus responsible for the outbreak of COVID-19, a disease that has spread to over 100 countries and, as of the 26th July 2020, has infected over 16 million people. Despite the urgent need to find effective therapeutics, research on SARS-CoV-2 has been affected by a lack of suitable animal models. To facilitate the development of medical approaches and novel treatments, we compared the ACE2 receptor, and TMPRSS2 and Furin proteases usage of the SARS-CoV-2 Spike glycoprotein in human and in a panel of animal models, i.e. guinea pig, dog, cat, rat, rabbit, ferret, mouse, hamster and macaque. Here we showed that ACE2, but not TMPRSS2 or Furin, has a higher level of sequence variability in the Spike protein interaction surface, which greatly influences Spike protein binding mode. Using molecular docking simulations we compared the SARS-CoV and SARS-CoV-2 Spike proteins in complex with the ACE2 receptor and showed that the SARS-CoV-2 Spike glycoprotein is compatible to bind the human ACE2 with high specificity. In contrast, TMPRSS2 and Furin are sufficiently similar in the considered hosts not to drive susceptibility differences. Computational analysis of binding modes and protein contacts indicates that macaque, ferrets and hamster are the most suitable models for the study of inhibitory antibodies and small molecules targeting the SARS-CoV-2 Spike protein interaction with ACE2. Since TMPRSS2 and Furin are similar across species, our data also suggest that transgenic animal models expressing human ACE2, such as the hACE2 transgenic mouse, are also likely to be useful models for studies investigating viral entry.

The COVID-19 pandemic, caused by the SARS-CoV-2 coronavirus, attacks various organs but most destructively the lung. It has been shown that SARS-CoV-2 entry into lung cells requires two host cell surface proteins: ACE2 and TMPRSS2. Downregulation of one or both of these is thus a potential therapeutic approach for COVID-19. TMPRSS2 is a known target of the androgen receptor, a ligand-activated transcription factor; activation of the androgen receptor increases TMPRSS2 levels in various tissues, most notably the prostate. We show here that treatment with the antiandrogen enzalutamide – a well-tolerated drug widely used in advanced prostate cancer – reduces TMPRSS2 levels in human lung cells. Further, enzalutamide treatment of mice dramatically decreased Tmprss2 levels in the lung. In support of this new experimental data, analysis of existing datasets shows striking co- expression of AR and TMPRSS2, including in specific lung cell types that are targeted by SARS- CoV-2. Together, the data presented provides strong evidence to support clinical trials to assess the efficacy of antiandrogens as a treatment option for COVID-19.

The SARS-CoV-2 coronavirus is the cause of the COVID-19 pandemic. Entry of the virus into host cells, most destructively lung cells, requires two host cell surface proteins, ACE2 and TMPRSS2, downregulation of which is thus a potential therapeutic approach for COVID-19. Both of these cell surface proteins are steroid regulated: TMPRSS2 is a well-characterised androgen-regulated target in prostate cancer. Analysis of sequencing data shows co-expression of the androgen receptor (AR) and TMPRSS2 in key human lung cell types that are targeted by SARS- CoV-2. We show that treatment with antiandrogens such as enzalutamide (a well-tolerated drug widely used in advanced prostate cancer) significantly reduces TMPRSS2 levels in human lung cells and in vivo in mouse lung. We demonstrate that AR binding in the region of the TMPRSS2 gene differs between lung and prostate, identifying distinct regulatory regions. Together, the data and evidence presented supports clinical trials to assess the efficacy of antiandrogens as a treatment option for COVID-19.

Mesenchymal stem cells (MSCs) are part of the tumour microenvironment and have been implicated in tumour progression. We found the number of MSCs significantly increased in tumour-burdened mice driven by Fas-threshold signalling. Consequently, MSCs lacking Fas lost their ability to induce metastasis development in a pancreatic cancer model. Mixing of MSCs with pancreatic cancer cells led to sustained production of the pro-metastatic cytokines CCL2 and IL6 by the stem cells. The levels of these cytokines depended on the number of MSCs, linking Fas-mediated MSC-proliferation to their capacity to promote tumour progression. Furthermore, we discovered that CCL2 and IL6 were induced by pancreatic cancer cell-derived IL1. Analysis of patient transcriptomic data revealed that high FasL expression correlates with high levels of MSC markers as well as increased IL6 and CCL2 in pancreatic tumours. Moreover, both FasL and CCL2 are linked to elevated levels of markers specific for monocytes known to possess further pro-metastatic activities. These results confirm our experimental findings of a FasL-MSC-IL1-CCL2/IL6 axis in pancreatic cancer and highlight the role MSCs play in tumour progression.

Cell therapy is a promising new treatment option for cancer. In particular, mesenchymal stem cells (MSCs) have shown potential in delivering therapeutic genes in various tumour models and are now on the verge of being tested in the clinic. A number of therapeutic genes have been examined in this context, including the death ligand TRAIL. For cell therapy, it can be used in its natural form as a full-length and membrane-bound protein (FL-TRAIL) or as an engineered version commonly referred to as soluble TRAIL (sTRAIL). As to which is more therapeutically efficacious, contradicting results have been reported. We discovered that MSCs producing sTRAIL have significantly higher apoptosis-inducing activity than cells expressing FL-TRAIL and found that FL-TRAIL, in contrast to sTRAIL, is not secreted. We also demonstrated that TRAIL does induce the expression of pro-metastatic cytokines in prostate cancer cells, but that this effect could be overcome through combination with an AKT inhibitor. Thus, a combination consisting of small-molecule drugs specifically targeting tumour cells in combination with MSC.sTRAIL, not only provides a way of sensitising cancer cells to TRAIL, but also reduces the issue of side-effect-causing cytokine production. This therapeutic strategy therefore represents a novel targeted treatment option for advanced prostate cancer and other difficult to treat tumours.

Activating transcription factor-2 (ATF-2) has been implicated as a tumour suppressor in breast cancer (BC). c-JUN N-terminal kinase (JNK) and p38 MAPK phosphorylate ATF-2 within the activation domain (AD), which is required for its transcriptional activity. To date, the role of ATF-2 in determining response to endocrine therapy has not been explored. Effects of ATF-2 loss in the oestrogen receptor (ER)-positive luminal BC cell line MCF7 were explored, as well as its role in response to tamoxifen treatment. Genome-wide chromatin binding patterns of ATF-2 when phosphorylated within the AD in MCF-7 cells were determined using ChIP-seq. The expression of ATF-2 and phosphorylated ATF-2 (pATF-2-Thr71) was determined in a series of 1,650 BC patients and correlated with clinico-pathological features and clinical outcome. Loss of ATF-2 diminished the growth-inhibitory effects of tamoxifen, while tamoxifen treatment induced ATF-2 phosphorylation within the AD, to regulate the expression of a set of 227 genes for proximal phospho-ATF-2 binding, involved in cell development, assembly and survival. Low expression of both ATF-2 and pATF-2-Thr71 was significantly associated with aggressive pathological features. Furthermore, pATF-2 was associated with both p-p38 and pJNK1/2 (

Breast cancer (BC) is traditionally viewed as an oestrogen-dependent disease in which the androgen receptor (AR) is inhibitory, counteracting the oncogenic activity of oestrogen receptor α (ERα (ESR1)). Most probably as a result of this crosstalk, the AR has prognostic value in ER-positive disease, with AR positivity reported to correlate with a better prognosis. Activation of the AR pathway has been previously used as a therapeutic strategy to treat BC, but its usage declined following the introduction of the anti-oestrogen tamoxifen. More recently, it has been demonstrated that a subset of triple-negative BCs (molecular apocrine) are dependent upon androgen signalling for growth and therapies that inhibit androgen signalling, currently used for the treatment of prostate cancer, e.g. the antiandrogen bicalutamide and the CYP17 inhibitor abiraterone acetate are undergoing clinical trials to investigate their efficacy in this BC subtype. This review summarises the current knowledge of AR activity in BC.

The development and growth of prostate cancer is dependent on androgens; thus, the identification of androgen-regulated genes in prostate cancer cells is vital for defining the mechanisms of prostate cancer development and progression and developing new markers and targets for prostate cancer treatment. GlycineN-methyltransferase (GNMT) is aS-adenosylmethionine-dependent methyltransferase that has been recently identified as a novel androgen-regulated gene in prostate cancer cells. Although the importance of this protein in prostate cancer progression has been extensively addressed, little is known about the mechanism of its androgen regulation. Here, we show that GNMT expression is stimulated by androgen in androgen receptor (AR) expressing cells and that the stimulation occurs at the mRNA and protein levels. We have identified an androgen response element within the first exon of theGNMTgene and demonstrated that AR binds to this elementin vitroandin vivo. Together, these studies identify GNMT as a direct transcriptional target of the AR. As this is an evolutionarily conserved regulatory element, this highlights androgen regulation as an important feature of GNMT regulation.

Progression of prostate cancer is highly dependent upon the androgen receptor pathway, such that knowledge of androgen-regulated proteins is vital to understand and combat this disease. Using a proteomic screen, we found the RNA-binding protein FUS/TLS (Fused in Ewing's Sarcoma/Translocated in Liposarcoma) to be downregulated in response to androgen. FUS has recently been shown to be recruited by noncoding RNAs to the regulatory regions of target genes such as cyclin D1, in which it represses transcription by disrupting complex formation. Here we show that FUS has some characteristics of a putative tumor suppressor, as its overexpression promoted growth inhibition and apoptosis of prostate cancer cells, whereas its knockdown increased cell proliferation. This effect was reproducible in vivo, such that increasing FUS levels in tumor xenografts led to dramatic tumor regression. Furthermore, FUS promoted conditions that favored cell-cycle arrest by reducing the levels of proliferative factors such as cyclin D1 and Cdk6 and by increasing levels of the antiproliferative Cdk inhibitor p27. Immunohistochemical analysis revealed that FUS expression is inversely correlated with Gleason grade, demonstrating that patients with high levels of FUS survived longer and were less likely to have bone metastases, suggesting that loss of FUS expression may contribute to cancer progression. Taken together, our results address the question of how androgens regulate cell-cycle progression, by demonstrating that FUS is a key link between androgen receptor signaling and cell-cycle progression in prostate cancer. Cancer Res; 71(3); 914–24. ©2010 AACR.

Prostate tumour growth is almost always dependent upon the androgen receptor pathway and hence therapies aimed at blocking this signalling axis are useful tools in the management of this disease. Unfortunately such therapies invariably fail; and the tumour progresses to an “androgen-independent” stage. In such cases androgen receptor expression is almost always maintained and much evidence exists to suggest that it may still be driving growth. One mechanism by which the receptor is thought to remain active is mutation. This review summarises the present data on androgen receptor mutations in prostate cancer, and how such substitutions offer a growth advantage by affecting cofactor interactions or by reducing ligand specificity. Such alterations appear to have a subsequent effect upon gene expression suggesting that tumours may “behave” differently dependent upon the ligand promoting growth and if a mutation is present.

Current therapies for prostate cancer include antiandrogens, inhibitory ligands of the androgen receptor, which repress androgen-stimulated growth. These include the selective androgen receptor modulators cyproterone acetate and hydroxyflutamide and the complete antagonist bicalutamide. Their activity is partly dictated by the presence of androgen receptor mutations, which are commonly detected in patients who relapse while receiving antiandrogens, i.e. in castrate-resistant prostate cancer. To characterize the early proteomic response to these antiandrogens we used the LNCaP prostate cancer cell line, which harbors the androgen receptor mutation most commonly detected in castrate-resistant tumors (T877A), analyzing alterations in the proteome, and comparing these to the effect of these therapeutics upon androgen receptor activity and cell proliferation. The majority are regulated post-transcriptionally, possibly via nongenomic androgen receptor signaling. Differences detected between the exposure groups demonstrate subtle changes in the biological response to each specific ligand, suggesting a spectrum of agonistic and antagonistic effects dependent on the ligand used. Analysis of the crystal structures of the AR in the presence of cyproterone acetate, hydroxyflutamide, and DHT identified important differences in the orientation of key residues located in the AF-2 and BF-3 protein interaction surfaces. This further implies that although there is commonality in the growth responses between androgens and those antiandrogens that stimulate growth in the presence of a mutation, there may also be influential differences in the growth pathways stimulated by the different ligands. This therefore has implications for prostate cancer treatment because tumors may respond differently dependent upon which mutation is present and which ligand is activating growth, also for the design of selective androgen receptor modulators, which aim to elicit differential proteomic responses dependent upon cellular context.

Anti-androgens used in prostate cancer therapy inhibit AR (androgen receptor) activity via largely unknown mechanisms. Although initially successful in most cases, they eventually fail and the disease progresses. We need to elucidate how anti-androgens work to understand why they fail, and prolong their effects or design further therapies. Using a cellular model, we found different anti-androgens have diverse effects on subcellular localization of AR, revealing that they work via different mechanisms and suggesting that an informed sequential treatment regime may benefit patients. In the presence of the anti-androgens bicalutamide and hydroxyflutamide, a significant proportion of the AR is translocated to the nucleus but remains inactive. Receptor inhibition under these conditions is likely to involve recruitment of co-repressor proteins, which interact with antagonist-occupied receptor but inhibit receptor-dependent transcription. Which co-repressors are required in vivo for AR repression by anti-androgens is not clear, but one candidate is the Notch effector Hey1. This inhibits ligand-dependent activity of the AR but not other steroid receptors. Further, it is excluded from the nucleus in most human prostate cancers, suggesting that abnormal subcellular distribution of co-repressors may contribute to the aberrant hormonal responses observed in prostate cancer. A decrease in co-repressor function is one possible explanation for the development of anti-androgen-resistant prostate cancer, and this suggests that it may not occur at the gross level of protein expression.

Prohibitin (PHB) is a cell cycle regulatory protein, known to repress E2F1-mediated gene activation via recruitment of transcriptional regulatory factors such as retinoblastoma and histone deacetylase 1 (HDAC1). We previously identified PHB as a target protein of androgen signaling in prostate cancer cells and showed that downregulation of PHB is required for androgen-induced cell cycle entry in these cells. We now present evidence that PHB, which has 54% homology at the protein level to the oestrogen receptor corepressor REA (repressor of oestrogen receptor activity), can repress androgen receptor (AR)-mediated transcription and androgen-dependent cell growth. Depletion of endogenous PHB resulted in an increase in expression of the androgen-regulated prostate-specific antigen gene. The repression appears to be specific to androgen and closely related receptors, as it is also evident for the glucocorticoid and progesterone, but not oestrogen, receptors. In spite of interaction of PHB with HDAC1, HDAC activity is not required for this repression. Although AR and PHB could be co-immunoprecipitated, no direct interaction was detectable, suggesting that PHB forms part of a repressive complex with the AR. Competition with the co-activator SRC1 further suggests that formation of a complex with AR, PHB and other cofactors is the mechanism by which repression is achieved. It appears then that repression of AR activity is one mechanism by which PHB inhibits androgen-dependent growth of prostate cells. Further, this study implies that the AR itself could, by mediating downregulation of a corepressor, be involved in the progression of prostate tumours to the hormone refractory stage.

Prostate cancer is an androgen receptor (AR)‐dependent malignancy at initiation and progression, therefore hormone therapy is the primary line of systemic treatment. Despite initial disease regression, tumours inevitably recur and progress to an advanced castration‐resistant state a major feature of which is metastasis to the bone. Up‐regulation of AR cofactors and chaperones that overcome low hormone conditions to maintain basal AR activity has been postulated as a mechanism of therapy relapse. p23, an essential component of the apo‐AR complex, acts also after ligand binding to increase AR transcriptional activity and target gene expression, partly by increasing chromatin‐loaded holo‐receptor‐complexes. Immunohistochemical studies have demonstrated increased p23 expression in advanced prostate cancer. Here, we further characterise p23 roles in AR signalling and show that it modulates cytosolic AR levels in the absence of hormone, confirming a chaperoning function in the aporeceptor complex and suggesting p23 upregulates AR signalling at multiple stages. Moreover, p23 protein levels significantly increased upon treatment with not only androgen but also clinically relevant anti‐androgens. This was in contrast to the HSP90 inhibitor 17‐AAG, which did not modulate expression of the cochaperone – important given the HSP90‐independent roles we and others have previously described for p23. Further, we demonstrate p23 is implicated in prostate cancer cell motility and in acquisition of invasiveness capacity through the expression of specific genes known to participate in cancer progression. This may drive metastatic processes in vivo since analysis of prostate tumour biopsies revealed that high nuclear p23 significantly correlated with shorter survival times and with development of metastases in patients with lower grade tumours. We propose that increased p23 expression may allow cells to acquire a more aggressive phenotype, contributing to disease progression, and that p23 is a plausible secondary target in combination with HSP90 inhibition as a potential therapy for advanced prostate cancer., Highlights We report a novel function for p23 in prostate cancer progression.p23 protein levels increase upon treatment with androgens and anti‐androgens.p23 promotes prostate cancer cell motility and acquisition of invasiveness.High nuclear p23 significantly correlates with shorter survival times in patients.p23 may reprogram gene expression profile into a more metastatic pattern.

Prostate cancer, the most common cancer of western men, requires new biomarkers, especially given that the benefits of PSA testing remain uncertain. Nucleic acids can now be accurately and sensitively detected in human blood. Over the last decade, investigations into utility of circulating cell-free miRNA, DNA and mRNA as novel biomarkers have expanded exponentially. In the near future, they may be routinely used to accurately diagnose cancers, stratify indolent from aggressive disease and inform treatment decisions. However, advancement of such tests into clinical settings is hampered by technical problems with assay specificity and sensitivity, and small study sizes. This review highlights the different forms of circulating nucleic acids and those that show the most potential as viable biomarkers for prostate cancer.

Prostate tumor growth initially depends on androgens, which act via the androgen receptor (AR). Despite androgen ablation therapy, tumors eventually progress to a castrate-resistant stage in which the AR remains active. The mechanisms are poorly understood but it may be that changes in levels or activity of AR coregulators affect trafficking and activation of the receptor. A key stage in AR signaling occurs in the cytoplasm, where unliganded receptor is associated with the heat shock protein (HSP)90 foldosome complex. p23, a key component of this complex, is best characterized as a cochaperone for HSP90 but also has HSP90-independent activity and has been reported as having differential effects on the activity of different steroid receptors. Here we report that p23 increases activity of the AR, and this appears to involve steps both in the cytoplasm (increasing ligand-binding capacity, possibly via direct interaction with AR) and the nucleus (enhancing AR occupancy at target promoters). We show, for the first time, that AR and p23 can interact, perhaps directly, when HSP90 is not present in the same complex. The effects of p23 on AR activity are at least partly HSP90 independent because a mutant form of p23, unable to bind HSP90, nevertheless increases AR activity. In human prostate tumors, nuclear p23 was higher in malignant prostate cells compared with benign/normal cells, supporting the utility of p23 as a therapeutic target in prostate cancer.

Androgen receptor (AR) is a major therapeutic target that plays pivotal roles in prostate cancer (PCa) and androgen insensitivity syndromes. We previously proposed that compounds recruited to ligand-binding domain (LBD) surfaces could regulate AR activity in hormone-refractory PCa and discovered several surface modulators of AR function. Surprisingly, the most effective compounds bound preferentially to a surface of unknown function [binding function 3 (BF-3)] instead of the coactivator-binding site [activation function 2 (AF-2)]. Different BF-3 mutations have been identified in PCa or androgen insensitivity syndrome patients, and they can strongly affect AR activity. Further, comparison of AR x-ray structures with and without bound ligands at BF-3 and AF-2 showed structural coupling between both pockets. Here, we combine experimental evidence and molecular dynamic simulations to investigate whether BF-3 mutations affect AR LBD function and dynamics possibly via allosteric conversation between surface sites. Our data indicate that AF-2 conformation is indeed closely coupled to BF-3 and provide mechanistic proof of their structural interconnection. BF-3 mutations may function as allosteric elicitors, probably shifting the AR LBD conformational ensemble toward conformations that alter AF-2 propensity to reorganize into subpockets that accommodate N-terminal domain and coactivator peptides. The induced conformation may result in either increased or decreased AR activity. Activating BF-3 mutations also favor the formation of another pocket (BF-4) in the vicinity of AF-2 and BF-3, which we also previously identified as a hot spot for a small compound. We discuss the possibility that BF-3 may be a protein-docking site that binds to the N-terminal domain and corepressors. AR surface sites are attractive pharmacological targets to develop allosteric modulators that might be alternative lead compounds for drug design.

Prostate cancers, initially responsive to anti-androgen therapies, often advance to a hormone-refractory “castrate resistant” stage (CRPC). However the androgen receptor (AR) pathway remains active and key for cell growth and gene expression within tumours, even in the apparent absence of hormone. Proposed mechanisms to explain progression, including AR amplification/mutation, are insufficient to completely explain CRPC and possible roles of AR cofactors such as prohibitin are poorly understood. We investigated whether prohibitin loss could sensitise prostate cancer cells and tumours to adrenal gland-derived androgens which persist even after androgen ablation, hence contribute to development of CRPC. Using a pair of prostate cancer cell lines, inducibly expressing ectopic cDNA or RNAi for PHB, responses to different androgens and hormone concentrations were studied both in vitro and in vivo. PHB was found at the promoters of several genes, both AR and non AR-regulated, and knockdown increased histone acetylation at these promoters. Further, PHB knockdown increased rate of AR ligand-induced chromatin binding, and binding rate and occupancy of AR upon the PSA promoter. This resulted in increased cell growth and AR activity in response to all androgens, including promoting a response to the weaker adrenal androgens previously absent at physiological concentrations. In vivo this had functional consequences such that PHB knockdown resulted in androstenedione being sufficient to promote tumour growth, under conditions mimicking those in patients undergoing androgen ablation therapy. We conclude that reduction in prohibitin levels is sufficient to lower the threshold of AR activity in vitro and in vivo; this may be via a general increase in histone acetylation that could potentially affect signalling by other transcription factors. Prohibitin loss may provide a mechanism for progression to CRPC by sensitizing prostate cancer cells to “castrate” conditions i.e. low levels of testicular androgens in the continued presence of weak adrenal and dietary androgens.

Cyclic acid anhydrides possessing a lipid chain have been shown to be a new class of non-peptidic inhibitors of geranylgeranyl protein-transferase type I (GGPTase-I).

Some pyrazino[1,2-a]indole-1,4-diones, structurally simplified analogues of the natural mycotoxin gliotoxin, have been synthesised and investigated as inhibitors of prenyltransferases; one compound, 3-acetylthio-9-methoxy-2-methyl-2,3-dihydropyrazino[1,2-a]indole-1,4-dione 10 shows slightly greater selectivity (8-fold) for geranylgeranyltransferase type I (GGTase I) than gliotoxin tself.

Some pyrazino[1,2-a]indole-1,4-diones, structurally simplified analogues of the natural mycotoxin gliotoxin, have been synthesised and investigated as inhibitors of prenyltransferases; one compound, 3-acetylthio-9-methoxy-2-methyl-2,3-dihydropyrazino[1,2-a]indole-1,4-dione 10 shows slightly greater selectivity (8-fold) for geranylgeranyltransferase type I (GGTase I) than gliotoxin itself.

Cyclic acid anhydrides possessing a lipid chain have been shown to be a new class of non-peptidic inhibitors of geranylgeranyl protein-transferase type I (GGPTase-I).

Background The RNA binding protein FUS is a multifunctional protein that has recently been demonstrated to be directed to target genes via non-coding RNA, where it represses transcription via disruption of the transcriptional complex. We have shown that FUS is an important regulator of prostate cancer cell growth; FUS over-expression causes G1 arrest, promotes apoptosis and blocks proliferation in vitro and in vivo, whereas knock-down increases proliferation. This regulation appears to be, at least in part, via direct regulation of cyclin D1. Further, analysis of patient samples demonstrated that FUS levels are inversely correlated with Gleason grade and directly correlated with survival and the presence of bone metastases. We hypothesised that FUS is a predictive and/or prognostic marker for hormone-dependent cancers. Materials and Methods: FUS expression in breast cancer was analysed in silico and using Western blotting and immunohistochemistry on a tumour microarray. FUS levels were knocked down by siRNA in MCF-7 cells and chemosensitivity analysed using caspase 3/7 assays. Results: Analysis of the Finak dataset (2008) demonstrated that FUS expression is significantly lower in breast cancer compared to normal breast. Immunohistochemistry revealed that FUS expression is also inversely correlated with grade. Importantly, FUS expression was also correlated with survival (Kaplan-Meier Plotter); significantly longer relapse free survival was seen in patients whose tumours had high levels of FUS. FUS expression also appears to correlate with therapy response since analysis of the Chang dataset (2003) revealed that FUS levels are significantly lower in patients resistant to Docetaxel and we found FUS levels to be lower in the tamoxifen resistant MCF-7 cell line (MCF-7 TAMR) compared to the parental line. We therefore hypothesised that loss of FUS expression may be driving therapy failure. In support of this, reducing FUS expression in the parental MCF7 line resulted in a significant reduction in docetaxel-induced Caspase 3/7 activity, suggesting that FUS is a regulator of chemosensitivity. Discussion: FUS may be an important regulator of breast cancer progression since its levels are inversely correlated with grade and directly correlated with relapse free survival. Further, lower FUS expression is also correlated with reduced hormone therapy and chemo-sensitivity. Since knock-down of FUS reduced MCF-7 chemosensitivity, we believe that the observed changes in FUS expression are, in part, driving therapy failure and hence propose that FUS is a novel target and biomarker for breast cancer. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-06-06.

$('.header-date').hide();$('#titleAuthors').hide(); $('#abstractHeader').hide(); Greg N. Brooke 1 , 2 , Sue M. Powell 1 , Derek N. Lavery 1 , Jonathan Waxman 1 , Laki Buluwela 1 , Simak Ali 1 and Charlotte L. Bevan 1 1 Department of Surgery and Cancer, Imperial Centre for Translational and Experimental Medicine, Imperial College London, W12 0NN, UK. 2 School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, UK. Correspondence: Charlotte Bevan, Email: charlotte.bevan@imperial.ac.uk Keywords: androgen receptor, prostate cancer, anti-androgen, castrate resistant prostate cancer Received : September 05, 2013 Accepted : November 20, 2013 Published : January 21, 2014 ABSTRACT Prostate cancer growth is dependent upon the Androgen Receptor (AR) pathway, hence therapies for this disease often target this signalling axis. Such therapies are successful in the majority of patients but invariably fail after a median of 2 years and tumours progress to a castrate resistant stage (CRPC). Much evidence exists to suggest that the AR remains key to CRPC growth and hence remains a valid therapeutic target. Here we describe a novel method to inhibit AR activity, consisting of an interaction motif, that binds to the AR ligand-binding domain, fused to repression domains. These ‘engineered repressors’ are potent inhibitors of AR activity and prostate cancer cell growth and importantly inhibit the AR under circumstances in which conventional therapies would be predicted to fail, such as AR mutation and altered cofactor levels.