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Cellular heterogeneity within the tissue microenvironment may underlie chemotherapeutic resistance and response, enabling tumor evolution; however, this heterogeneity it is difficult to characterize. Here, we present a new approach-pathology-guided micropunching (PGM)-that enables identification and characterization of heterogeneous foci identified in viable human and animal model tissue slices. This technique consists of live-cell tissue labeling using fluorescent antibodies/small molecules to identify heterogeneous foci (e.g., immune infiltrates or cells with high levels of reactive oxygen species) in viable tissues, coupled with a micropunch step to isolate cells from these heterogeneous foci for downstream molecular or vital functional analysis. Micropunches obtained from epithelial or stromal fibroblast foci in human prostate tissue show 6- to 12-fold enrichment in transcripts specific for EpCam/cytokeratin 8 and vimentin/a-smooth muscle actin/integrin 1-α, respectively. Transcriptional enrichment efficiency agrees with epithelial and stromal laser capture microdissection samples isolated from human prostate. Micropunched foci show a loss of cellular viability in the periphery, but centrally localized cells retained viability before and after dissociation and grew out in culture.

Objective: To evaluate the accuracy, readability, understandability, and actionability of Internet patient education materials (PEM) about transrectal ultrasound-guided prostate biopsy., Methods: A comprehensive Internet search was performed to find PEM with pre- or postbiopsy instructions. PEM that were duplicates, government affiliated, international, or video based were excluded. Biopsy instructions were evaluated for accuracy and presence of essential topics. Readability was assessed via word count and Flesch-Kincaid Grade Level. Understandability and actionability were measured using the Patient Education Materials Assessment Tool (PEMAT). Effects of authorship and geographical variation were determined using Fischer exact and Kruskal-Wallis tests., Results: We identified 148 unique PEM. Only 31 (21%) sites adhered to the recommended <8th grade reading level. Most PEM did not contain recommended graphics (14%), checklists (2%), or summaries (6%). The PEMAT understandability score for academic PEM was higher than private (P = .02) and unaffiliated PEM (P = .01). No websites had inaccurate content. Only 2 PEM sites (1%) included all essential content (stop anticoagulants, antibiotics, need for urinalysis, biopsy pain, when to resume activity, and bleeding complications). Few significant differences based on geographic region were observed for word count, readability, PEMAT scores, or content., Conclusion: Transrectal ultrasound-guided prostate biopsy PEM adhere poorly to guidelines for easy-to-understand materials. Most PEM lack vital information and are written at a reading level that is too complex for patient comprehension. The urology community can construct better websites by consulting PEM advisory materials and providing nontechnical language, figures, and specific instructions., (Published by Elsevier Inc.)

Loss of imprinting (LOI) is an epigenetic event that relaxes an allele-specific restriction on gene expression. One gene that experiences LOI is the paracrine insulin-like growth factor IGF2 , which occurs commonly in human prostate tissues during aging and tumorigenesis. However, the relationship between IGF2 LOI and prostate tumorigenesis has not been established functionally. In this study, we created a mouse model with CTCF-binding site mutations at the Igf2-H19 imprint control region that abolishes CTCF insulator activity, resulting in biallelic Igf2 expression that mimics increased levels seen with aging-induced LOI. We found that Igf2 LOI increased the prevalence and severity of prostatic intraepithelial neoplasia (PIN), a premalignant lesion. Engineering Nkx3.1 deficiency into our model increased the frequency of PIN lesions in an additive fashion. Prostates harboring LOI displayed increased MAPK signaling and epithelial proliferation. In human prostate tissue arrays, we documented a positive correlation in benign tissues of IGF2 levels with phospho-ERK and phospho-AKT levels. Overall, our results establish that Igf2 LOI is sufficient on its own to increase rates of neoplastic development in the prostate by upregulating critical cancer-associated signaling pathways. Cancer Res; 77(19); 5236-47. ©2017 AACR ., (©2017 American Association for Cancer Research.)

No guidelines currently exist that address the need for rebiopsy in patients with a negative diagnosis of prostate cancer on initial biopsy sample analysis. Accurate diagnosis of prostate cancer in these patients is often complicated by continued elevation of serum PSA levels that are suggestive of prostate cancer, resulting in a distinct management challenge. Following negative initial findings of biopsy sample analysis, total serum PSA levels and serum PSA kinetics are ineffective indicators of a need for a repeat biopsy; therefore, patients suspected of having prostate cancer might undergo several unnecessary biopsy procedures. Several alternative strategies exist for identifying men who might be at risk of prostate cancer despite negative findings of biopsy sample analysis. Use of other serum PSA-related measurements enables more sensitive and specific diagnosis and can be combined with knowledge of clinicopathological features to improve outcomes. Other options include the FDA-approved Progensa(®) test and prostate imaging using MRI. Newer tissue-based assays that measure methylation changes in normal prostate tissue are currently being developed. A cost-effective strategy is proposed in order to address this challenging clinical scenario, and potential directions of future studies in this area are also described.

Androgen receptor (AR) signaling initiates mouse prostate development by stimulating prostate ductal bud formation and specifying bud patterns. Curiously, however, prostatic bud initiation lags behind the onset of gonadal testosterone synthesis by about three days. This study's objective was to test the hypothesis that DNA methylation controls the timing and scope of prostate ductal development by regulating Ar expression in the urogenital sinus (UGS) from which the prostate derives. We determined that Ar DNA methylation decreases in UGS mesenchyme during prostate bud formation in vivo and that this change correlates with decreased DNA methyltransferase expression in the same cell population during the same time period. To examine the role of DNA methylation in prostate development, fetal UGSs were grown in serum-free medium and 5 alpha dihydrotestosterone (DHT) and the DNA methylation inhibitor 5'-aza-2'-deoxycytidine (5AzadC) were introduced into the medium at specific times. As a measure of prostate development, in situ hybridization was used to visualize and count Nkx3-1 mRNA positive prostatic buds. We determined that inhibiting DNA methylation when prostatic buds are being specified, accelerates the onset of prostatic bud development, increases bud number, and sensitizes the budding response to androgens. Inhibition of DNA methylation also reduces Ar DNA methylation in UGS explants and increases Ar mRNA and protein in UGS mesenchyme and epithelium. Together, these results support a novel mechanism whereby Ar DNA methylation regulates UGS androgen sensitivity to control the rate and number of prostatic buds formed, thereby establishing a developmental checkpoint., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Prostate cancer (PCa) is typically found as a multifocal disease suggesting the potential for molecular defects within the morphologically normal tissue. The frequency and spatial extent of DNA methylation changes encompassing a potential field defect are unknown. A comparison of non-tumor-associated (NTA) prostate to histologically indistinguishable tumor-associated (TA) prostate tissues detected a distinct profile of DNA methylation alterations (0.2%) using genome-wide DNA arrays based on the Encyclopedia of DNA Elements 18 sequence that tile both gene-rich and poor regions. Hypomethylation (87%) occurred more frequently than hypermethylation (13%). Several of the most significantly altered loci (CAV1, EVX1, MCF2L, and FGF1) were then used as probes to map the extent of these DNA methylation changes in normal tissues from prostates containing cancer. In TA tissues, the extent of methylation was similar both adjacent (2 mm) and at a distance (>1 cm) from tumor foci. These loci were also able to distinguish NTA from TA tissues in a validation set of patient samples. These mapping studies indicate that a spatially widespread epigenetic defect occurs in the peripheral prostate tissues of men who have PCa that may be useful in the detection of this disease.

Increased oxidative stress and concordant DNA methylation changes are found during aging and in many malignant processes including prostate cancer. Increased oxidative stress has been shown to inhibit DNA methyltransferase in in vitro assays, but whether this occurs in vivo is unknown. To generate increased oxidative stress we utilized mice containing mutations in the CuZnSOD (Sod1) gene, a major superoxide dismutase in mammals. Increased 8-hydroxy-2'-deoxyguanosine, an adduct indicating oxidative damage, was found in liver and prostate tissues at 2 and 12 mo Sod1 (+/-) mice compared to controls. Prostate tissues from Sod1 (+/-) mice demonstrated decreased weight at 2 mo compared to controls, but this difference was not significant at 12 mo. Histologic changes were not seen. Global DNA methylation was significantly decreased at 2 mo in the prostate in Sod1 (+/-) mice. 11p15 containing the epigenetically modulated insulin-like growth factor 2 (Igf2) and H19 genes, both which display oncogenic functions, may be particularly sensitive to oxidative stress. CpG island methylation at an intergenic CTCF binding site and the Igf2 P3 promoter was decreased in Sod1 mutants compared to controls. This is the first in vivo study to show that a deficiency of Sod1 leads to a decrease in DNA methylation. These studies indicate that increased oxidative stress, a factor implicated in neoplasia, can induce DNA hypomethylation in prostate tissues.

Loss of imprinting (LOI) is an epigenetic alteration involving loss of parental origin-specific expression at normally imprinted genes. A LOI for Igf2, a paracrine growth factor, is important in cancer progression. Epigenetic modifications may be altered by environmental factors. However, is not known whether changes in imprinting occur with aging in prostate and other tissues susceptible to cancer development. We found a LOI for Igf2 occurs specifically in the mouse prostate associated with increased Igf2 expression during aging. In older animals, expression of the chromatin insulator protein CTCF and its binding to the Igf2-H19 imprint control region was reduced. Forced down-regulation of CTCF leads to Igf2 LOI. We further show that Igf2 LOI occurs with aging in histologically normal human prostate tissues and that this epigenetic alteration was more extensive in men with associated cancer. This finding may contribute to a postulated field of cancer susceptibility that occurs with aging. Moreover, Igf2 LOI may serve as a marker for the presence of prostate cancer.

Background: Folate and methyl-group deficiency has been linked to prostate cancer susceptibility, yet the mechanisms underlying these observations are incompletely understood. The region of the genome containing the imprinted genes insulin-like growth factor 2 (Igf2) and H19, both of which display oncogenic functions, may be particularly sensitive to environmental influences., Methods: To determine whether a methyl-deficient diet impacts epigenetic controls at the Igf2-H19 locus, we placed C57BL/6 mice containing a polymorphism at the imprinted Igf2-H19 locus on a choline and methionine deficient (CMD) diet. We interrogated this locus for expression and epigenetic changes in prostate tissues., Results: A significant increase in both Igf2 and H19 expression was found in CMD prostate tissues compared to controls. These expression changes were reversible with shorter exposure to the CMD diet. Chromatin immunoprecipitation (ChIP) revealed significant decreases in repressive histone modifications (dimethyl-H3K9) within the H19 promoter, as well as Igf2 P2 and P3 promoters. DNA methylation within these promoters was not altered. No significant change in Igf2 or H19 imprinting was observed., Conclusions: These findings highlight the plasticity of the epigenome in an epithelial organ vulnerable to neoplastic change. They further suggest that chromatin modifications are more susceptible to methyl-deficient diets than DNA methylation at this locus., (Copyright (c) 2008 Wiley-Liss, Inc.)

Background: Proliferative inflammatory atrophy (PIA) of the prostate is a common histological finding that has been postulated to be associated with prostate cancer. We examine PIA lesions for the expression of p16/CDKN2, a cyclin-dependent kinase inhibitor frequently altered in prostate cancer., Methods: Within tissues from two independent patient populations ("Test Set" and "Validation Set") undergoing radical prostatectomy, PIA lesions were identified and subjected to immunohistochemical staining for p16. Atrophic epithelial cells and regional normal epithelium were scored for the extent of p16 staining. In the Test Set, staining was also performed for the proliferation marker Ki-67. Double label immunofluorescence was employed to co-localize staining for p16 and Ki-67., Results: p16 expression was elevated in PIA in both data sets compared to normal epithelium (mean percent of cells staining positive in PIA = 11.1%-16.2%; mean percent of cells staining positive in normal = 0.6%-1.3%) (P = 0.0001). As expected from prior studies, the mean Ki-67 index was higher in PIA lesions (mean 8.2% staining positive) versus normal epithelium (mean 1.9% staining positive) (P = 0.0001), and the extent of staining for p16 correlated with Ki-67 (r = 0.7, P < 0.0001). However, co-localization immunofluorescent studies did not demonstrate staining for nuclear p16 and Ki-67 in the same cells., Conclusions: Increased p16 expression accompanies increased cell proliferation in PIA lesions of the prostate. Yet, on the individual cell level, the upregulation of p16 in PIA lesions appears to represent a non-proliferative stress response, possibly to oxidative damage.

Cdc37 is a co-chaperone protein that recruits several immature client kinases to Hsp90 for proper folding. Cdc37 up-regulation is a common early event in localized human prostate cancer. Although targeted overexpression in mice leads to prostate epithelial cell hyperplasia, the effect of Cdc37 dysregulation in human prostate cells is unclear. In this study, we examine the role of Cdc37 in the growth regulation of normal prostate epithelial cells using a unique human model system. We demonstrate that Cdc37 overexpression drives proliferation, whereas loss of Cdc37 function arrests growth and leads to apoptosis. With increased Cdc37 expression, molecular analysis of Cdc37 client pathways demonstrates enhanced Raf-1 activity, greater Cdk4 levels, and reduced expression of the cyclin-dependent kinase inhibitor p16/CDKN2. To further investigate these downstream pathways, enhanced Raf-1 or Cdk4 activities were selectively induced in human prostate epithelial cells. Raf-1 activation inhibited proliferation and generated an enlarged, flattened morphology. Induction of Cdk4 activity using cyclin D1 overexpression, however, was sufficient to promote proliferation. These data indicate that Cdc37 induces proliferation and is critical for survival in human prostate epithelial cells. These alterations in cell division and survival may be important in the development and progression of early prostate cancer.

Cellular senescence forms a barrier that inhibits the acquisition of an immortal phenotype, a critical feature in tumorigenesis. The inactivation of multiple pathways that positively regulate senescence are required for immortalization. To identify these pathways in an unbiased manner, we performed DNA microarray analyses to assess the expression of 20,000 genes in human prostate epithelial cells (HPECs) passaged to senescence. These gene expression patterns were then compared with those of HPECs immortalized with the human Papillomavirus 16 E7 oncoprotein. Senescent cells display gene expression patterns that reflect their nonproliferative, differentiated phenotype and express secretory proteases and extracellular matrix components. A comparison of genes transcriptionally up-regulated in senescence to those in which expression is significantly down-regulated in immortalized HPECs identified three genes: the chemokine BRAK, DOC1, and a member of the insulin-like growth factor axis, IGFBP-3. Expression of these genes is found to be uniformly lost in human prostate cancer cell lines and xenografts, and previously, their inactivation was documented in tumor samples. Thus, these genes may function in novel pathways that regulate senescence and are inactivated during immortalization. These changes may be critical not only in allowing cells to bypass senescence in vitro but in the progression of prostate cancer in vivo.

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.

The cell cycle regulatory genes p16/CDKN2 and RB are frequently deleted in prostate cancers. In this study, we examined the role of alterations in p16 and pRb during growth, senescence, and immortalization in vitro of human prostate epithelial cells (HPECs). HPECs are established from normal prostate tissues and cultured on collagen-coated dishes. Our results show that p16 is reproducibly elevated at senescence in HPECs. HPECs are immortalized using human papilloma virus 16 E6 and/or E7 as molecular tools to inactivate p53 and/or pRb, respectively. Immortalization occurs infrequently in this system and only after a latent period during which additional genetic/epigenetic changes are thought to occur. Notably, all of the E6-immortalized HPEC lines but none of the E7 lines show inactivation of p16/CDKN2 (by deletion, methylation, or mutation) in association with immortalization. In contrast, E7 lines, in which pRb function is abrogated by E7 binding, retain the high levels of p16 observed at senescence. Thus, all lines show either a p16 or pRb inactivation. Analysis of six independent lines from metastatic prostate cancers reveals a similar loss of either p16 or pRb. Comparative genomic hybridization of HPECs shows that gains of chromosomes 5q, 8q, and 20 are nonrandomly associated with bypassing senescence (probability = 0.95). These results suggest that high levels of the cyclin-dependent kinase inhibitor p16 mediate senescence G1 arrest in HPECs and that bypassing this block by a p16/pRb pathway alteration is required for immortalization in vitro and possibly tumorigenesis in vivo. Our results further indicate that inactivation of the p16/pRb pathway alone is not sufficient to immortalize HPECs and that additional genetic alterations are required for this process.

P-Cadherin is a member of the cadherin family of cell surface glycoproteins that mediate Ca2+-dependent cell-cell adhesion and is expressed in a differential fashion in normal epithelial tissues. The expression of P-cadherin in human prostate cancer development has not been investigated previously. By immunohistochemistry, we show that P-cadherin expression is restricted to the cell-cell border of basal epithelial cells in 30 normal prostate samples. This staining is down-regulated in prostatic intraepithelial neoplasia and is absent in all 25 of the well to poorly differentiated prostate cancer specimens analyzed. To examine potential P-cadherin-regulatory elements, we sequenced the 5'-flanking region of this gene. Similar to the mouse gene, the human P-cadherin promoter is TATA-less, contains an Sp-1 binding site and, analogous to the human E-cadherin sequence, demonstrates a GC-rich region characteristic of a CpG island. Cytosine methylation of this region occurs in P-cadherin-negative prostate cancer cell lines but not in cell lines expressing this gene. In vivo, a lack of expression in 12 clinical prostate cancer specimens is not associated with methylation of the P-cadherin promoter. These results demonstrate that the expression of the basal cell marker P-cadherin is lost in prostate cancer development and that in vivo mechanisms other than cytosine methylation regulate this consistent loss of expression.

In most tissues, the insulin-like growth factor II gene (IGF-II) demonstrates imprinting, being expressed exclusively from the paternal allele. Recently, a loss of IGF-II imprinting (i.e., biallelic expression) has been found in sporadic Wilm's tumors and lung carcinomas, and this molecular event may contribute to the pathogenesis of these tumors. Here, we report that in prostates removed at radical surgery for localized adenocarcinoma, both the cancer and the associated normal peripheral zone tissue have a pronounced biallelic expression of the IGF-II gene. However, this pattern of gene expression is uncommon in periurethral samples of benign prostatic hyperplasia (BPH) from the same specimens. We analyzed the status of genomic imprinting at the IGF-II locus in prostate specimens removed for carcinoma using an ApaI polymorphism in the 3' untranslated exon of the IGF-II gene. First-strand cDNA synthesis and subsequent PCR amplification were performed on 13 of 35 radical prostatectomy specimens found to be informative for analysis of allele-specific expression. Biallelic expression for IGF-II RNA was demonstrated in 10 (83%) of 12 tumor samples and 8 (73%) of 11 matched peripheral zone prostate samples but in only 2 (18%) of 11 BPH samples. RNA transcripts were readily demonstrated by Northern blot analysis, and differences in expression were not noted among normal, BPH, and tumor prostate tissues. In situ hybridization revealed production of IGF-II by both the epithelium and stroma. The finding of a frequent biallelic expression of IGF-II in peripheral prostate specimens suggests a regional pattern of IGF-II gene regulation exists in prostate tissue. We hypothesize that this tissue-specific pattern of gene expression may participate in the marked predilection of peripheral prostatic tissue for the development of carcinogenesis.