Compartmentalization of androgen receptors at endogenous genes in living cells.

A wide range of nuclear proteins are involved in the spatio-temporal organization of the genome through diverse biological processes such as gene transcription and DNA replication. Upon stimulation by testosterone and translocation to the nucleus, multiple androgen receptors (ARs) accumulate in microscopically discernable foci which are irregularly distributed in the nucleus. Here, we investigated the formation and physical nature of these foci, by combining novel fluorescent labeling techniques to visualize a defined chromatin locus of AR-regulated genes-PTPRN2 or BANP-simultaneously with either AR foci or individual AR molecules. Quantitative colocalization analysis showed evidence of AR foci formation induced by R1881 at both PTPRN2 and BANP loci. Furthermore, single-particle tracking (SPT) revealed three distinct subdiffusive fractional Brownian motion (fBm) states: immobilized ARs were observed near the labeled genes likely as a consequence of DNA-binding, while the intermediate confined state showed a similar spatial behavior but with larger displacements, suggesting compartmentalization by liquid-liquid phase separation (LLPS), while freely mobile ARs were diffusing in the nuclear environment. All together, we show for the first time in living cells the presence of AR-regulated genes in AR foci.

Transposable Elements Are Co-opted as Oncogenic Regulatory Elements by Lineage-Specific Transcription Factors in Prostate Cancer.

Transposable elements hold regulatory functions that impact cell fate determination by controlling gene expression. However, little is known about the transcriptional machinery engaged at transposable elements in pluripotent and mature versus oncogenic cell states. Through positional analysis over repetitive DNA sequences of H3K27ac chromatin immunoprecipitation sequencing data from 32 normal cell states, we report pluripotent/stem and mature cell state-specific "regulatory transposable elements." Pluripotent/stem elements are binding sites for pluripotency factors (e.g., NANOG, SOX2, OCT4). Mature cell elements are docking sites for lineage-specific transcription factors, including AR and FOXA1 in prostate epithelium. Expanding the analysis to prostate tumors, we identify a subset of regulatory transposable elements shared with pluripotent/stem cells, including Tigger3a. Using chromatin editing technology, we show how such elements promote prostate cancer growth by regulating AR transcriptional activity. Collectively, our results suggest that oncogenesis arises from lineage-specific transcription factors hijacking pluripotent/stem cell regulatory transposable elements. SIGNIFICANCE: We show that oncogenesis relies on co-opting transposable elements from pluripotent stem cells as regulatory elements altering the recruitment of lineage-specific transcription factors. We further discover how co-option is dependent on active chromatin states with important implications for developing treatment options against drivers of oncogenesis across the repetitive DNA. This article is featured in Selected Articles from This Issue, p. 2293.

Grade Group 1 Prostate Cancers Exhibit Tumor-defining Androgen Receptor-driven Programs.

Grade group 1 (GG1) primary prostate cancers with a pathologic Gleason score of 6 are considered indolent and generally not associated with fatal outcomes, so treatment is not indicated for most cases. These low-grade cancers have an overall negligible risk of locoregional progression and metastasis to distant organs, which is why there is an ongoing debate about whether these lesions should be reclassified as "noncancerous". However, the underlying molecular activity of key disease drivers, such as the androgen receptor (AR), have thus far not been thoroughly characterized in low-grade tumors. Therefore, we set out to delineate the AR chromatin-binding landscape in low-grade GG1 prostate cancers to gain insights into whether these AR-driven programs are actually tumor-specific or are normal prostate epithelium-like. These analyses showed that GG1 tumors do not harbor a distinct AR cistrome and, similar to higher-grade cancers, AR preferentially binds to tumor-defining cis-regulatory elements. Furthermore, the enhancer activity of these regions and the expression of their respective target genes were not significantly different in GG1 tumors. From an epigenetic perspective, this finding supports the cancer designation currently given to these low-grade tumors and clearly distinguishes them from noncancerous benign tissue. PATIENT SUMMARY: We characterized the molecular activity of the androgen receptor protein, which drives prostate cancer disease, in low-grade tumors. Our results show that these tumors are true cancers and are clearly separate from benign prostate tissue despite their low clinical aggressiveness.

Radiogenomics Analysis Linking Multiparametric MRI and Transcriptomics in Prostate Cancer.

Prostate cancer (PCa) is a highly prevalent cancer type with a heterogeneous prognosis. An accurate assessment of tumor aggressiveness can pave the way for tailored treatment strategies, potentially leading to better outcomes. While tumor aggressiveness is typically assessed based on invasive methods (e.g., biopsy), radiogenomics, combining diagnostic imaging with genomic information can help uncover aggressive (imaging) phenotypes, which in turn can provide non-invasive advice on individualized treatment regimens. In this study, we carried out a parallel analysis on both imaging and transcriptomics data in order to identify features associated with clinically significant PCa (defined as an ISUP grade ≥ 3), subsequently evaluating the correlation between them. Textural imaging features were extracted from multi-parametric MRI sequences (T2W, DWI, and DCE) and combined with DCE-derived parametric pharmacokinetic maps obtained using magnetic resonance dispersion imaging (MRDI). A transcriptomic analysis was performed to derive functional features on transcription factors (TFs), and pathway activity from RNA sequencing data, here referred to as transcriptomic features. For both the imaging and transcriptomic features, different machine learning models were separately trained and optimized to classify tumors in either clinically insignificant or significant PCa. These models were validated in an independent cohort and model performance was used to isolate a subset of relevant imaging and transcriptomic features to be further investigated. A final set of 31 imaging features was correlated to 33 transcriptomic features obtained on the same tumors. Five significant correlations (p < 0.05) were found, of which, three had moderate strength (|r| ≥ 0.5). The strongest significant correlations were seen between a perfusion-based imaging feature-MRDI A median-and the activities of the TFs STAT6 (-0.64) and TFAP2A (-0.50). A higher-order T2W textural feature was also significantly correlated to the activity of the TF STAT6 (-0.58). STAT6 plays an important role in controlling cell proliferation and migration. Loss of the AP2alpha protein expression, quantified by TFAP2A, has been strongly associated with aggressiveness and progression in PCa. According to our findings, a combination of texture features extracted from T2W and DCE, as well as perfusion-based pharmacokinetic features, can be considered for the prediction of clinically significant PCa, with the pharmacokinetic MRDI A feature being the most correlated with the underlying transcriptomic information. These results highlight a link between quantitative imaging features and the underlying transcriptomic landscape of prostate tumors.

Enhancer profiling identifies epigenetic markers of endocrine resistance and reveals therapeutic options for metastatic castration-resistant prostate cancer patients.

Androgen Receptor (AR) signaling inhibitors, including enzalutamide, are treatment options for patients with metastatic castration-resistant prostate cancer (mCRPC), but resistance inevitably develops. Using metastatic samples from a prospective phase II clinical trial, we epigenetically profiled enhancer/promoter activities with H3K27ac chromatin immunoprecipitation followed by sequencing, before and after AR-targeted therapy. We identified a distinct subset of H3K27ac-differentially marked regions that associated with treatment responsiveness. These data were successfully validated in mCRPC patient-derived xenograft models (PDX). In silico analyses revealed HDAC3 as a critical factor that can drive resistance to hormonal interventions, which we validated in vitro . Using cell lines and mCRPC PDX tumors in vitro , we identified drug-drug synergy between enzalutamide and the pan-HDAC inhibitor vorinostat, providing therapeutic proof-of-concept. These findings demonstrate rationale for new therapeutic strategies using a combination of AR and HDAC inhibitors to improve patient outcome in advanced stages of mCRPC.

Genetic determinants of chromatin reveal prostate cancer risk mediated by context-dependent gene regulation.

Many genetic variants affect disease risk by altering context-dependent gene regulation. Such variants are difficult to study mechanistically using current methods that link genetic variation to steady-state gene expression levels, such as expression quantitative trait loci (eQTLs). To address this challenge, we developed the cistrome-wide association study (CWAS), a framework for identifying genotypic and allele-specific effects on chromatin that are also associated with disease. In prostate cancer, CWAS identified regulatory elements and androgen receptor-binding sites that explained the association at 52 of 98 known prostate cancer risk loci and discovered 17 additional risk loci. CWAS implicated key developmental transcription factors in prostate cancer risk that are overlooked by eQTL-based approaches due to context-dependent gene regulation. We experimentally validated associations and demonstrated the extensibility of CWAS to additional epigenomic datasets and phenotypes, including response to prostate cancer treatment. CWAS is a powerful and biologically interpretable paradigm for studying variants that influence traits by affecting transcriptional regulation.

Drug-Induced Epigenomic Plasticity Reprograms Circadian Rhythm Regulation to Drive Prostate Cancer toward Androgen Independence.

In prostate cancer, androgen receptor (AR)-targeting agents are very effective in various disease stages. However, therapy resistance inevitably occurs, and little is known about how tumor cells adapt to bypass AR suppression. Here, we performed integrative multiomics analyses on tissues isolated before and after 3 months of AR-targeting enzalutamide monotherapy from patients with high-risk prostate cancer enrolled in a neoadjuvant clinical trial. Transcriptomic analyses demonstrated that AR inhibition drove tumors toward a neuroendocrine-like disease state. Additionally, epigenomic profiling revealed massive enzalutamide-induced reprogramming of pioneer factor FOXA1 from inactive chromatin sites toward active cis-regulatory elements that dictate prosurvival signals. Notably, treatment-induced FOXA1 sites were enriched for the circadian clock component ARNTL. Posttreatment ARNTL levels were associated with patients' clinical outcomes, and ARNTL knockout strongly decreased prostate cancer cell growth. Our data highlight a remarkable cistromic plasticity of FOXA1 following AR-targeted therapy and revealed an acquired dependency on the circadian regulator ARNTL, a novel candidate therapeutic target. SIGNIFICANCE: Understanding how prostate cancers adapt to AR-targeted interventions is critical for identifying novel drug targets to improve the clinical management of treatment-resistant disease. Our study revealed an enzalutamide-induced epigenomic plasticity toward prosurvival signaling and uncovered the circadian regulator ARNTL as an acquired vulnerability after AR inhibition, presenting a novel lead for therapeutic development. See related commentary by Zhang et al., p. 2017. This article is highlighted in the In This Issue feature, p. 2007.

Unexpected gene activation following CRISPR-Cas9-mediated genome editing.

The discovery of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and its development as a genome editing tool has revolutionized the field of molecular biology. In the DNA damage field, CRISPR has brought an alternative to induce endogenous double-strand breaks (DSBs) at desired genomic locations and study the DNA damage response and its consequences. Many systems for sgRNA delivery have been reported in order to efficiently generate this DSB, including lentiviral vectors. However, some of the consequences of these systems are not yet well understood. Here, we report that lentiviral-based sgRNA vectors can integrate into the endogenous genomic target location, leading to undesired activation of the target gene. By generating a DSB in the regulatory region of the ABCB1 gene using a lentiviral sgRNA vector, we can induce the formation of Taxol-resistant colonies. We show that these colonies upregulate ABCB1 via integration of the EEF1A1 and the U6 promoters from the sgRNA vector. We believe that this is an unreported CRISPR/Cas9 on-target effect that researchers need to be aware of when using lentiviral vectors for genome editing.

Reduced NCOR2 expression accelerates androgen deprivation therapy failure in prostate cancer.

This study addresses the roles of nuclear receptor corepressor 2 (NCOR2) in prostate cancer (PC) progression in response to androgen deprivation therapy (ADT). Reduced NCOR2 expression significantly associates with shorter disease-free survival in patients with PC receiving adjuvant ADT. Utilizing the CWR22 xenograft model, we demonstrate that stably reduced NCOR2 expression accelerates disease recurrence following ADT, associates with gene expression patterns that include neuroendocrine features, and induces DNA hypermethylation. Stably reduced NCOR2 expression in isogenic LNCaP (androgen-sensitive) and LNCaP-C4-2 (androgen-independent) cells revealed that NCOR2 reduction phenocopies the impact of androgen treatment and induces global DNA hypermethylation patterns. NCOR2 genomic binding is greatest in LNCaP-C4-2 cells and most clearly associates with forkhead box (FOX) transcription factor FOXA1 binding. NCOR2 binding significantly associates with transcriptional regulation most when in active enhancer regions. These studies reveal robust roles for NCOR2 in regulating the PC transcriptome and epigenome and underscore recent mutational studies linking NCOR2 loss of function to PC disease progression.

An androgen receptor switch underlies lineage infidelity in treatment-resistant prostate cancer.

Cancers adapt to increasingly potent targeted therapies by reprogramming their phenotype. Here we investigated such a phenomenon in prostate cancer, in which tumours can escape epithelial lineage confinement and transition to a high-plasticity state as an adaptive response to potent androgen receptor (AR) antagonism. We found that AR activity can be maintained as tumours adopt alternative lineage identities, with changes in chromatin architecture guiding AR transcriptional rerouting. The epigenetic regulator enhancer of zeste homologue 2 (EZH2) co-occupies the reprogrammed AR cistrome to transcriptionally modulate stem cell and neuronal gene networks-granting privileges associated with both fates. This function of EZH2 was associated with T350 phosphorylation and establishment of a non-canonical polycomb subcomplex. Our study provides mechanistic insights into the plasticity of the lineage-infidelity state governed by AR reprogramming that enabled us to redirect cell fate by modulating EZH2 and AR, highlighting the clinical potential of reversing resistance phenotypes.

A kinome-centered CRISPR-Cas9 screen identifies activated BRAF to modulate enzalutamide resistance with potential therapeutic implications in BRAF-mutated prostate cancer.

Resistance to drugs targeting the androgen receptor (AR) signaling axis remains an important challenge in the treatment of prostate cancer patients. Activation of alternative growth pathways is one mechanism used by cancer cells to proliferate despite treatment, conferring drug resistance. Through a kinome-centered CRISPR-Cas9 screen in CWR-R1 prostate cancer cells, we identified activated BRAF signaling as a determinant for enzalutamide resistance. Combined pharmaceutical targeting of AR and MAPK signaling resulted in strong synergistic inhibition of cell proliferation. The association between BRAF activation and enzalutamide resistance was confirmed in two metastatic prostate cancer patients harboring activating mutations in the BRAF gene, as both patients were unresponsive to enzalutamide. Our findings suggest that co-targeting of the MAPK and AR pathways may be effective in patients with an activated MAPK pathway, particularly in patients harboring oncogenic BRAF mutations. These results warrant further investigation of the response to AR inhibitors in BRAF-mutated prostate tumors in clinical settings.

Functional mapping of androgen receptor enhancer activity.

BACKGROUND: Androgen receptor (AR) is critical to the initiation, growth, and progression of prostate cancer. Once activated, the AR binds to cis-regulatory enhancer elements on DNA that drive gene expression. Yet, there are 10-100× more binding sites than differentially expressed genes. It is unclear how or if these excess binding sites impact gene transcription. RESULTS: To characterize the regulatory logic of AR-mediated transcription, we generated a locus-specific map of enhancer activity by functionally testing all common clinical AR binding sites with Self-Transcribing Active Regulatory Regions sequencing (STARRseq). Only 7% of AR binding sites displayed androgen-dependent enhancer activity. Instead, the vast majority of AR binding sites were either inactive or constitutively active enhancers. These annotations strongly correlated with enhancer-associated features of both in vitro cell lines and clinical prostate cancer samples. Evaluating the effect of each enhancer class on transcription, we found that AR-regulated enhancers frequently interact with promoters and form central chromosomal loops that are required for transcription. Somatic mutations of these critical AR-regulated enhancers often impact enhancer activity. CONCLUSIONS: Using a functional map of AR enhancer activity, we demonstrated that AR-regulated enhancers act as a regulatory hub that increases interactions with other AR binding sites and gene promoters.

Dual functions of SPOP and ERG dictate androgen therapy responses in prostate cancer.

Driver genes with a mutually exclusive mutation pattern across tumor genomes are thought to have overlapping roles in tumorigenesis. In contrast, we show here that mutually exclusive prostate cancer driver alterations involving the ERG transcription factor and the ubiquitin ligase adaptor SPOP are synthetic sick. At the molecular level, the incompatible cancer pathways are driven by opposing functions in SPOP. ERG upregulates wild type SPOP to dampen androgen receptor (AR) signaling and sustain ERG activity through degradation of the bromodomain histone reader ZMYND11. Conversely, SPOP-mutant tumors stabilize ZMYND11 to repress ERG-function and enable oncogenic androgen receptor signaling. This dichotomy regulates the response to therapeutic interventions in the AR pathway. While mutant SPOP renders tumor cells susceptible to androgen deprivation therapies, ERG promotes sensitivity to high-dose androgen therapy and pharmacological inhibition of wild type SPOP. More generally, these results define a distinct class of antagonistic cancer drivers and a blueprint toward their therapeutic exploitation.

Androgen modulation of XBP1 is functionally driving part of the AR transcriptional program.

Prostate cancer development and progression is largely dependent on androgen receptor (AR) signaling. AR is a hormone-dependent transcription factor, which binds to thousands of sites throughout the human genome to regulate expression of directly responsive genes, including pro-survival genes that enable tumor cells to cope with increased cellular stress. ERN1 and XBP1 - two key players of the unfolded protein response (UPR) - are among such stress-associated genes. Here, we show that XBP1 levels in primary prostate cancer are associated with biochemical recurrence in five independent cohorts. Patients who received AR-targeted therapies had significantly lower XBP1 expression, whereas expression of the active form of XBP1 (XBP1s) was elevated. In vitro results show that AR-induced ERN1 expression led to increased XBP1s mRNA and protein levels. Furthermore, ChIP-seq analysis revealed that XBP1s binds enhancers upon stress stimuli regulating genes involved in UPR processes, eIF2 signaling and protein ubiquitination. We further demonstrate genomic overlap of AR- and XBP1s-binding sites, suggesting genomic conversion of the two signaling cascades. Transcriptomic effects of XBP1 were further studied by knockdown experiments, which lead to decreased expression of androgen-responsive genes and UPR genes. These results suggest a two-step mechanism of gene regulation, which involves androgen-induced expression of ERN1, thereby enhancing XBP1 splicing and transcriptional activity. This signaling cascade may prepare the cells for the increased protein folding, mRNA decay and translation that accompanies AR-regulated tumor cell proliferation.

Optimized ChIP-seq method facilitates transcription factor profiling in human tumors.

Chromatin immunoprecipitation (ChIP)-seq analyses of transcription factors in clinical specimens are challenging due to the technical limitations and low quantities of starting material, often resulting in low enrichments and poor signal-to-noise ratio. Here, we present an optimized protocol for transcription factor ChIP-seq analyses in human tissue, yielding an ∼100% success rate for all transcription factors analyzed. As proof of concept and to illustrate general applicability of the approach, human tissue from the breast, prostate, and endometrial cancers were analyzed. In addition to standard formaldehyde fixation, disuccinimidyl glutarate was included in the procedure, greatly increasing data quality. To illustrate the sensitivity of the optimized protocol, we provide high-quality ChIP-seq data for three independent factors (AR, FOXA1, and H3K27ac) from a single core needle prostate cancer biopsy specimen. In summary, double-cross-linking strongly improved transcription factor ChIP-seq quality on human tumor samples, further facilitating and enhancing translational research on limited amounts of tissue.

Enzalutamide therapy for advanced prostate cancer: efficacy, resistance and beyond

The androgen receptor drives the growth of metastatic castration-resistant prostate cancer. This has led to the development of multiple novel drugs targeting this hormone-regulated transcription factor, such as enzalutamide ­ a potent androgen receptor antagonist. Despite the plethora of possible treatment options, the absolute survival benefit of each treatment separately is limited to a few months. Therefore, current research efforts are directed to determine the optimal sequence of therapies, discover novel drugs effective in metastatic castration-resistant prostate cancer and define patient subpopulations that ultimately benefit from these treatments. Molecular studies provide evidence on which pathways mediate treatment resistance and may lead to improved treatment for metastatic castration-resistant prostate cancer. This review provides, firstly a concise overview of the clinical development, use and effectiveness of enzalutamide in the treatment of advanced prostate cancer, secondly it describes translational research addressing enzalutamide response vs resistance and lastly highlights novel potential treatment strategies in the enzalutamide-resistant setting.

Mps1 inhibitors synergise with low doses of taxanes in promoting tumour cell death by enhancement of errors in cell division.

BACKGROUND: Chromosomal instability (CIN) is a common trait of cancer characterised by the continuous gain and loss of chromosomes during mitosis. Excessive levels of CIN can suppress tumour growth, providing a possible therapeutic strategy. The Mps1/TTK kinase has been one of the prime targets to explore this concept, and indeed Mps1 inhibitors synergise with the spindle poison docetaxel in inhibiting the growth of tumours in mice. METHODS: To investigate how the combination of docetaxel and a Mps1 inhibitor (Cpd-5) promote tumour cell death, we treated mice transplanted with BRCA1-/-;TP53-/- mammary tumours with docetaxel and/or Cpd-5. The tumours were analysed regarding their histopathology, chromosome segregation errors, copy number variations and cell death to understand the mechanism of action of the drug combination. RESULTS: The enhanced efficacy of combining an Mps1 inhibitor with clinically relevant doses of docetaxel is associated with an increase in multipolar anaphases, aberrant nuclear morphologies and cell death. Tumours treated with docetaxel and Cpd-5 displayed more genomic deviations, indicating that chromosome stability is affected mostly in the combinatorial treatment. CONCLUSIONS: Our study shows that the synergy between taxanes and Mps1 inhibitors depends on increased errors in cell division, allowing further optimisation of this treatment regimen for cancer therapy.

Identification and synthesis of new volatile molecules found in extracts obtained from distinct parts of cooked chicken.

Several chicken parts (skin, fat, juice) were cooked in different ways (roasting, simmering) and investigated separately for their volatile composition. In-depth GC/MS analysis of the separate fractions revealed several unknown molecules. Mass spectra interpretation allowed us to identify nine molecules for the first time in chicken, including cyclic aldehydes, cyclic ketones, and new δ-lactones containing an unsaturated linear chain. Identification was confirmed by chemical synthesis followed by comparison of the mass spectra and linear retention indices. The natural occurrence of five of these molecules is reported here for the first time in a natural product.