Prostate Cancer Epigenetic Plasticity and Enhancer Heterogeneity: Molecular Causes, Consequences and Clinical Implications.

Prostate cancer (PCa) proliferation is dictated by androgen receptor (AR) signaling, which regulates gene expression through cis-regulatory regions including proximal and distal enhancers. The repertoire of AR interactions at enhancers is dependent on tissue and cellular contexts and thus shape a spectrum of phenotypes through such epigenetic heterogeneity. Moreover, PCa is a multifocal disease and displays a high degree of intra- and inter-tumor heterogeneity, adding to the phenotypic complexity. It is increasingly becoming clear that PCa may be considered an epigenetic disease caused by various molecular causes with profound consequences and clinical implications which are underpinned by enhancer interaction heterogeneity.In this review, we provide a detailed overview of molecular interactors that affect prostate cancer epigenetic heterogeneity, such as coding and non-coding somatic variants, large scale structural variations, pioneer factor binding at enhancers and various contexts that influence enhancer engagement heterogeneity in PCa development and progression. Finally, we explore how the vast heterogeneity in epigenetic profiles identified in recent omics studies results in distinct genomic subtypes which predict disease progression and thus offer opportunities in biomarker discovery and further personalizing cancer treatment. As such, heterogeneous enhancer interactions take center stage in elucidating mechanisms of prostate cancer progression, patient prognostication, therapy discovery and overcoming acquired treatment resistance.

DNA-guided DNA cleavage at moderate temperatures by Clostridium butyricum Argonaute.

Prokaryotic Argonaute proteins (pAgos) constitute a diverse group of endonucleases of which some mediate host defense by utilizing small interfering DNA guides (siDNA) to cleave complementary invading DNA. This activity can be repurposed for programmable DNA cleavage. However, currently characterized DNA-cleaving pAgos require elevated temperatures (≥65°C) for their activity, making them less suitable for applications that require moderate temperatures, such as genome editing. Here, we report the functional and structural characterization of the siDNA-guided DNA-targeting pAgo from the mesophilic bacterium Clostridium butyricum (CbAgo). CbAgo displays a preference for siDNAs that have a deoxyadenosine at the 5'-end and thymidines at nucleotides 2-4. Furthermore, CbAgo mediates DNA-guided DNA cleavage of AT-rich double stranded DNA at moderate temperatures (37°C). This study demonstrates that certain pAgos are capable of programmable DNA cleavage at moderate temperatures and thereby expands the scope of the potential pAgo-based applications.

Agent-based modeling of the prostate tumor microenvironment uncovers spatial tumor growth constraints and immunomodulatory properties.

Inhibiting androgen receptor (AR) signaling through androgen deprivation therapy (ADT) reduces prostate cancer (PCa) growth in virtually all patients, but response may be temporary, in which case resistance develops, ultimately leading to lethal castration-resistant prostate cancer (CRPC). The tumor microenvironment (TME) plays an important role in the development and progression of PCa. In addition to tumor cells, TME-resident macrophages and fibroblasts express AR and are therefore also affected by ADT. However, the interplay of different TME cell types in the development of CRPC remains largely unexplored. To understand the complex stochastic nature of cell-cell interactions, we created a PCa-specific agent-based model (PCABM) based on in vitro cell proliferation data. PCa cells, fibroblasts, "pro-inflammatory" M1-like and "pro-tumor" M2-like polarized macrophages are modeled as agents from a simple set of validated base assumptions. PCABM allows us to simulate the effect of ADT on the interplay between various prostate TME cell types. The resulting in vitro growth patterns mimic human PCa. Our PCABM can effectively model hormonal perturbations by ADT, in which PCABM suggests that CRPC arises in clusters of resistant cells, as is observed in multifocal PCa. In addition, fibroblasts compete for cellular space in the TME while simultaneously creating niches for tumor cells to proliferate in. Finally, PCABM predicts that ADT has immunomodulatory effects on macrophages that may enhance tumor survival. Taken together, these results suggest that AR plays a critical role in the cellular interplay and stochastic interactions in the TME that influence tumor cell behavior and CRPC development.

Exploring the Onset and Progression of Prostate Cancer through a Multicellular Agent-based Model.

Over 10% of men will be diagnosed with prostate cancer during their lifetime. Arising from luminal cells of the prostatic acinus, prostate cancer is influenced by multiple cells in its microenvironment. To expand our knowledge and explore means to prevent and treat the disease, it is important to understand what drives the onset and early stages of prostate cancer. In this study, we developed an agent-based model of a prostatic acinus including its microenvironment, to allow for in silico studying of prostate cancer development. The model was based on prior reports and in-house data of tumor cells cocultured with cancer-associated fibroblasts (CAF) and protumor and/or antitumor macrophages. Growth patterns depicted by the model were pathologically validated on hematoxylin and eosin slide images of human prostate cancer specimens. We identified that stochasticity of interactions between macrophages and tumor cells at early stages strongly affect tumor development. In addition, we discovered that more systematic deviations in tumor development result from a combinatorial effect of the probability of acquiring mutations and the tumor-promoting abilities of CAFs and macrophages. In silico modeled tumors were then compared with 494 patients with cancer with matching characteristics, showing strong association between predicted tumor load and patients' clinical outcome. Our findings suggest that the likelihood of tumor formation depends on a combination of stochastic events and systematic characteristics. While stochasticity cannot be controlled, information on systematic effects may aid the development of prevention strategies tailored to the molecular characteristics of an individual patient. Significance: We developed a computational model to study which factors of the tumor microenvironment drive prostate cancer development, with potential to aid the development of new prevention strategies.

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.

Extensive androgen receptor enhancer heterogeneity in primary prostate cancers underlies transcriptional diversity and metastatic potential.

Androgen receptor (AR) drives prostate cancer (PCa) development and progression. AR chromatin binding profiles are highly plastic and form recurrent programmatic changes that differentiate disease stages, subtypes and patient outcomes. While prior studies focused on concordance between patient subgroups, inter-tumor heterogeneity of AR enhancer selectivity remains unexplored. Here we report high levels of AR chromatin binding heterogeneity in human primary prostate tumors, that overlap with heterogeneity observed in healthy prostate epithelium. Such heterogeneity has functional consequences, as somatic mutations converge on commonly-shared AR sites in primary over metastatic tissues. In contrast, less-frequently shared AR sites associate strongly with AR-driven gene expression, while such heterogeneous AR enhancer usage also distinguishes patients' outcome. These findings indicate that epigenetic heterogeneity in primary disease is directly informative for risk of biochemical relapse. Cumulatively, our results illustrate a high level of AR enhancer heterogeneity in primary PCa driving differential expression and clinical impact.

Oligometastatic Prostate Cancer: Results of a Dutch Multidisciplinary Consensus Meeting.

BACKGROUND: Oligometastatic prostate cancer (OMPC) is a heterogeneous disease state that is imperfectly understood, and its clinical implications are unclear. OBJECTIVE: To determine the consensus of a Dutch multidisciplinary expert panel on biological aspects, treatment goals, and management of OMPC in daily clinical practice. DESIGN, SETTING, AND PARTICIPANTS: The study comprised a modified Delphi method including an explorative survey with various statements and questions, followed by a consensus meeting to discuss and determine the agreement with revised statements and related items. The panel consisted of 34 Dutch representatives from urology, medical and radiation oncology, radiology, nuclear medicine, and basic research. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Agreement was determined with statements (five-point scale). Consensus was defined as ≥75% panel agreement with a statement. RESULTS AND LIMITATIONS: Consensus existed for 56% of statements. The panel agreed that OMPC comprises a limited metastatic spread in the hormone-sensitive setting, in both the synchronous and the metachronous presentation. Limited metastatic spread was believed to involve three to five metastases and a maximum of two organs. Prostate-specific membrane antigen positron emission tomography/computed tomography scan was currently perceived as the most accurate diagnostic imaging modality. Although there was a consensus that targeted treatment of all metastases in OMPC will delay further dissemination of the disease, opinions on specific treatment regimens were divided. Panel outcomes were limited by the lack of scientific evidence on OMPC. CONCLUSIONS: A multidisciplinary panel reached a consensus that OMPC is a specific disease state requiring a tailored treatment approach. OMPC registries and clinical studies should focus on both the biology and the clinical parameters in relation to optimal treatment strategies in synchronous and metachronous OMPC. PATIENT SUMMARY: A group of Dutch medical specialists agreed that prostate cancer patients having few metastases may benefit from a new therapeutic approach. Clinical studies need to determine which treatment is best for each specific situation.

Frequent clonal relations between metastases and non-index prostate cancer lesions.

Primary prostate cancer lesions are clonally heterogeneous and often arise independently. In contrast, metastases were reported to share a monoclonal background. Because prostate cancer mortality is the consequence of distant metastases, prevention of metastatic outgrowth by primary tumor ablation is the main focus of treatment for localized disease. Focal therapy is targeted ablation of the primary index lesion, but it is unclear whether remaining primary lesions metastasize at a later stage. In this study, we compared copy number aberration profiles of primary prostate cancer lesions with matching pelvic lymph node metastases of 30 patients to establish clonality between a lymph node metastasis and multiple primary lesions within the same patient. Interestingly, in 23.3% of the cases, the regional metastasis was not clonally linked to the index primary lesion. These findings suggest that focal ablation of only the index lesion is potentially an undertreatment of a significant proportion of prostate cancer patients.

Multiplex gene editing by CRISPR-Cpf1 using a single crRNA array.

Targeting of multiple genomic loci with Cas9 is limited by the need for multiple or large expression constructs. Here we show that the ability of Cpf1 to process its own CRISPR RNA (crRNA) can be used to simplify multiplexed genome editing. Using a single customized CRISPR array, we edit up to four genes in mammalian cells and three in the mouse brain, simultaneously.