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In this trial, the addition of six cycles of docetaxel to androgen-deprivation therapy resulted in longer median progression-free and overall survival than that with ADT alone in patients with metastatic prostate cancer. Regressions of metastatic prostate cancer were first documented in the 1940s and were achieved with surgical castration; subsequently, androgen-deprivation therapy (ADT) became the mainstay of therapy. 1 Attempts to improve the efficacy or decrease the treatment burden of ADT have included the use of antiandrogens alone, intermittent dosing of ADT, and the use of an antiandrogen combined with medical or surgical castration. 2 – 4 A meta-analysis revealed an increase in survival of 3 percentage points at 5 years with concurrent use of a nonsteroidal antiandrogen at the time of initiation of ADT. 2 However, resistance to ADT occurs in most patients, with the . . .

Androgen deprivation therapy (ADT) commonly leads to incomplete cell death and the fate of persistent cells involves, in part, a senescent phenotype. Senescence is terminal growth arrest in response to cell stress that is characterized by increased lysosomal-β-galactosidase (GLB1) the origin of senescence associated-β-gal activity (SA-β-gal). In the current study senescence is examined in vivo after ADT use in a neoadjuvant trial. Tissue microarrays were generated from prostate cancer specimens (n = 126) from a multicenter neoadjuvant ADT trial. Arrays were subjected to multiplexed immunofluorescent staining for GLB1, Ki67, cleaved caspase 3 (CC3) and E-cadherin. Automated quantitative imaging was performed using Vectra™ and expression correlated with clinicopathologic features. Tissue was analyzed from 59 patients treated with neoadjuvant ADT and 67 receiving no therapy preoperatively. Median follow-up was 85.3 mo and median ADT treatment was 5 mo. In PC treated with neoadjuvant ADT, GLB1 expression increased in intermediate Gleason score (GS 6-7; p = 0.001), but not high grade (GS 8-10) cancer. Significantly higher levels of GLB1 were seen in tissues undergoing neoadjuvant ADT longer than 5 months compared to untreated tissues (p = 0.002). In contrast, apoptosis significantly increased earlier (1-4 mo) after ADT treatment (p<0.5). Increased GLB1 after neoadjuvant ADT occurs primarily among more clinically favorable intermediate grade cancers and enrichment of the phenotype occurs in a temporally prolonged fashion. Senescence may explain the persistence of PCa cells after ADT. Given concerns for the detrimental longer term presence of senescent cells, targeting these cells for removal may improve outcomes.

The folate pathway plays a crucial role in the regeneration and repair of the adult CNS after injury. Here, we have shown in rodents that such repair occurs at least in part through DNA methylation. In animals with combined spinal cord and sciatic nerve injury, folate-mediated CNS axon regeneration was found to depend on injury-related induction of the high-affinity folate receptor 1 (Folr1). The activity of folate was dependent on its activation by the enzyme dihydrofolate reductase (Dhfr) and a functional methylation cycle. The effect of folate on the regeneration of afferent spinal neurons was biphasic and dose dependent and correlated closely over its dose range with global and gene-specific DNA methylation and with expression of both the folate receptor Folr1 and the de novo DNA methyltransferases. These data implicate an epigenetic mechanism in CNS repair. Folic acid and possibly other nontoxic dietary methyl donors may therefore be useful in clinical interventions to promote brain and spinal cord healing. If indeed the benefit of folate is mediated by epigenetic mechanisms that promote endogenous axonal regeneration, this provides possible avenues for new pharmacologic approaches to treating CNS injuries.

Downregulation of HLA class I (HLA-I) impairs immune recognition and surveillance in prostate cancer and may underlie the ineffectiveness of checkpoint blockade. However, the molecular mechanisms regulating HLA-I loss in prostate cancer have not been fully explored. Here, we conducted a comprehensive analysis of HLA-I genomic, epigenomic and gene expression alterations in primary and metastatic human prostate cancer. Loss of HLA-I gene expression was associated with repressive chromatin states including DNA methylation, histone H3 tri-methylation at lysine 27, and reduced chromatin accessibility. Pharmacological DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibition decreased DNA methylation and increased H3 lysine 27 acetylation and resulted in re-expression of HLA-I on the surface of tumor cells. Re-expression of HLA-I on LNCaP cells by DNMT and HDAC inhibition increased activation of co-cultured prostate specific membrane antigen (PSMA) 27-38 -specific CD8 + T-cells. HLA-I expression is epigenetically regulated by functionally reversible DNA methylation and chromatin modifications in human prostate cancer. Methylated HLA-I was detected in HLA-I low circulating tumor cells (CTCs), which may serve as a minimally invasive biomarker for identifying patients who would benefit from epigenetic targeted therapies. Loss of HLA-I gene expression in prostate cancer is associated with repressive chromatin states, which can be reversed by pharmacological DNMT and HDAC inhibition leading to increased activation of co-cultured tumor-specific CD8+ T-cells.

Genomic imprinting is the allele-specific expression of a gene based on parental origin. Loss of imprinting(LOI) of Insulin-like Growth Factor 2 (IGF2) during aging is important in tumorigenesis, yet the regulatory mechanisms driving this event are largely unknown. In this study oxidative stress, measured by increased NF-κB activity, induces LOI in both cancerous and noncancerous human prostate cells. Decreased expression of the enhancer-blocking element CCCTC-binding factor(CTCF) results in reduced binding of CTCF to the H19-ICR (imprint control region), a major factor in the allelic silencing of IGF2. This ICR then develops increased DNA methylation. Assays identify a recruitment of the canonical pathway proteins NF-κB p65 and p50 to the CTCF promoter associated with the co-repressor HDAC1 explaining gene repression. An IκBα super-repressor blocks oxidative stress-induced activation of NF-κB and IGF2 imprinting is maintained. In vivo experiments using IκBα mutant mice with continuous NF-κB activation demonstrate increased IGF2 LOI further confirming a central role for canonical NF-κB signaling. We conclude CTCF plays a central role in mediating the effects of NF-κB activation that result in altered imprinting both in vitro and in vivo. This novel finding connects inflammation found in aging prostate tissues with the altered epigenetic landscape.

Background To assess factors that can predict active surveillance (AS) failure on serial transrectal ultrasound guided biopsies in patients with low-risk prostate cancer. Methods We evaluated the records of 144 consecutive patients enrolled in AS between 2007 and 2014 at a single academic institution. Low risk inclusion criteria included PSA < 10 ng/ml, cT1c or cT2a, Grade Group (GG) 1, < 3 positive cores, and < 50% tumor in a single core with the majority having a PSA density of < 0.15. AS reclassification was defined as progression to GG ≥2, 3 or more cores, or core tumor volume ≥ 50%. Univariate and multivariate Cox proportional hazards regression analysis was used to determine predictors of reclassification and a match-pair analysis performed on a control group of patients choosing surgery. Results Inclusion criteria were met by 130 men with a median follow-up of 52 months. The reclassification or AS failure rate was 38.5%, with the majority 41/50 (82%) finding GG ≥ 2 cancer. Most patients had unilateral disease on diagnostic biopsy (94.6%), but 40.7% had bilateral cancer detected during follow-up. Men with bilateral detected tumor were more likely to ultimately fail AS than patients with unilateral tumors (HR 4.089; P  < 0.0001) and failed earlier with a reclassification-free survival of 32 vs 119 months respectively. In a matched-pair analysis using a population of 211 concurrent patients that chose radical prostatectomy rather than AS, 76% of patients with unilateral cancer on biopsy had bilateral cancer on final pathology. Conclusions The finding of bilateral prostate cancer on biopsy is associated with earlier AS reclassification. Finding bilateral disease may not represent disease progression, but rather enhanced detection of more extensive disease highlighting the importance of confirmatory biopsy.

The study of intercellular signalling networks requires organotypic microscale systems that facilitate the culture, conditioning and manipulation of cells. Here, we describe a reconfigurable microfluidic cell-culture system that facilitates the assembly of three-dimensional tissue models by stacking layers that contain preconditioned microenvironments. By using principles of open and suspended microfluidics, the Stacks system is easily assembled or disassembled to provide spatial and temporal manoeuvrability in two-dimensional and three-dimensional assays of multiple cell types, enabling the modelling of sequential paracrine-signalling events, such as tumour-cell-mediated differentiation of macrophages and macrophage-facilitated angiogenesis. We used Stacks to recapitulate the in vivo observation that different prostate cancer tissues polarize macrophages with distinct gene-expression profiles of pro-inflammatory and anti-inflammatory cytokines. Stacks also enabled us to show that these two types of macrophages signal distinctly to endothelial cells, leading to blood vessels with different morphologies. Our proof-of-concept experiments exemplify how Stacks can efficiently model multicellular interactions and highlight the importance of spatiotemporal specificity in intercellular signalling. A reconfigurable microfluidic cell-culture system that facilitates the assembly of 3D tissue models by stacking layers containing preconditioned microenvironments enables the modelling of the spatiotemporal dynamics of paracrine signalling.

Background The chromatin insulator CCCTC-binding factor (CTCF) displays tissue-specific DNA binding sites that regulate transcription and chromatin organization. Despite evidence linking CTCF to the protection of epigenetic states through barrier insulation, the impact of CTCF loss on genome-wide DNA methylation sites in human cancer remains undefined. Results Here, we demonstrate that prostate and breast cancers within The Cancer Genome Atlas (TCGA) exhibit frequent copy number loss of CTCF and that this loss is associated with increased DNA methylation events that occur preferentially at CTCF binding sites. CTCF sites differ among tumor types and result in tissue-specific methylation patterns with little overlap between breast and prostate cancers. DNA methylation and transcriptome profiling in vitro establish that forced downregulation of CTCF leads to spatially distinct DNA hypermethylation surrounding CTCF binding sites, loss of CTCF binding, and decreased gene expression that is also seen in human tumors. DNA methylation inhibition reverses loss of expression at these CTCF-regulated genes. Conclusion These findings establish CTCF loss as a major mediator in directing localized DNA hypermethylation events in a tissue-specific fashion and further support its role as a driver of the cancer phenotype.

Prostate Cancer (PC) is a disease with remarkable tumor heterogeneity that often manifests in significant intra-patient variability with regards to clinical outcomes and treatment response. Commonly available PC cell lines do not accurately reflect the complexity of this disease and there is critical need for development of new models to recapitulate the intricate hierarchy of tumor pathogenesis. In current study, we established ex vivo primary patient-derived cancer organoid (PDCO) cultures from prostatectomy specimens of patients with locally advanced PC. We then performed a comprehensive multi-parameter characterization of the cellular composition utilizing a novel approach for live-cell staining and direct imaging in the integrated microfluidic Stacks device. Using orthogonal flow cytometry analysis, we demonstrate that primary PDCOs maintain distinct subsets of epithelial cells throughout culture and that these cells conserve expression of androgen receptor (AR)-related elements. Furthermore, to confirm the tumor-origin of the PDCOs we have analyzed the expression of PC-associated epigenetic biomarkers including promoter methylation of the GSTP1, RASSF1 and APC and RARb genes by employing a novel microfluidic rare-event screening protocol. These results demonstrate that this ex vivo PDCO model recapitulates the complexity of the epithelial tumor microenvironment of multifocal PC using orthogonal analyses. Furthermore, we propose to leverage the Stacks microfluidic device as a high-throughput, translational platform to interrogate phenotypic and molecular endpoints with the capacity to incorporate a complex tumor microenvironment.

Cellular senescence has been proposed to be an in vitro and in vivo block that cells must overcome in order to immortalize and become tumorigenic. To characterize these pathways, we focused on changes in the cyclin-dependent kinase inhibitors and their binding partners that underlie the cell cycle arrest at senescence. As a model, we utilized normal human prostate epithelial cell (HPEC) and human uroepithelial cell (HUC) cultures. After 30-40 population doublings cells became growth-arrested in G0/1 with a threefold decrease in Cdk2-associated activity, a point defined as pre-senescence. Temporally following this growth arrest, the cells develop a senescence morphology and express senescence-associated beta-galactosidase (SA-beta-gal). Levels of p16(INK4a) and p57(KIP2) rise in HUCs during progressive passages, whereas only p16 increases in HPEC cultures. The induced expression of p57, similar to p16, produces a senescent-like phenotype. pRB, cyclin D, p19(INK4d) and p27(KIP1) decrease in both cell types. We find that p53, p21(CIP1) and p15(INK4b) are transiently elevated in HPECs and HUCs at the pre-senescent growth arrest, then return to low proliferating levels at terminal senescence. Analysis of p53, p21(CIP1), p15(INK4b), p16(INK4a), and p57(KIP2) reveals altered expression in immortalized, non-tumorigenic HPV16 E6 and E7 prostate lines and in tumorigenic prostate cancer cells. These results indicate: (i) the existence of a subset of growth inhibiting genes elevated at the onset of the senescence, (ii) a distinct class of genes involved in the maintenance of senescence, and (iii) the frequent inactivation of these pathways during immortalization.