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High-grade prostatic intraepithelial neoplasia (HGPIN) is a well-characterised precursor lesion in prostate cancer. The term atypical intraductal proliferations (AIP) describes lesions with features that are far too atypical to be considered HGPIN, yet insufficient to be diagnosed as intraductal carcinoma of the prostate (IDCP). Here, a panel of biomarkers was assessed to provide insights into the biological relationship between IDCP, HGPIN, and AIP and their relevance to current clinicopathological recommendations. Tissue samples from 86 patients with prostate cancer were assessed by routine haematoxylin and eosin staining and immunohistochemistry (IHC) with a biomarker panel (Appl1/Sortilin/Syndecan-1) and a PIN4 cocktail (34βE12+P63/P504S). Appl1 strongly labelled atypical secretory cells, effectively visualising intraductal lesions. Sortilin labelling was moderate-to-strong in > 70% of cases, while Syndecan-1 was moderate-to-strong in micropapillary HGPIN/AIP lesions (83% cases) versus flat/tufting HGPIN (≤ 20% cases). Distinct biomarker labelling patterns for atypical intraductal lesions of the prostate were observed, including early atypical changes (flat/tufting HGPIN) and more advanced atypical changes (micropapillary HGPIN/AIP). Furthermore, the biomarker panel may be used as a tool to overcome the diagnostic uncertainty surrounding AIP by supporting a definitive diagnosis of IDCP for such lesions displaying the same biomarker pattern as cribriform IDCP., (© 2023. The Author(s).)

Cocaine and amphetamine regulated transcript (CART) is a somatostatin-like polypeptide. CART has been localized in the CNS, hypothalamo-pituitary-adrenocortical (HPA) axis, pancreatic islets and enteric nervous system. We investigated the cellular localization of CART in normal human prostate, benign prostatic hyperplasia, prostatic intraepithelial neoplasia and acinar adenocarcinoma. CART was assessed using immunohistochemistry (IHC) and in situ hybridization (ISH), and its gene expression was identified by RTqPCR. We found cellular expression of CART in both normal prostatic luminal secretory epithelial cells neuroendocrine cells (NEC) of both ducts and acini. The cellular appearance indicated a cycle of neuropeptide synthesis and secretion as validated by ISH/IHC concordance. RTqPCR analysis also validated the immunohistochemical data and gene expression, which both indicated low to moderate expression in prostatic tissues. CART expression also was increased in both neuroendocrine and glandular epithelial cell populations from samples of benign prostatic hyperplasia as validated by IHC, ISH and RTqPCR. CART expression was markedly diminished and, in some cases, entirely absent in tissues of prostatic intraepithelial neoplasia and adenocarcinoma. Owing to loss of CART expression in adenocarcinoma and its increase in benign prostatic hyperplasia, CART may prove to be an important prostate marker.

Objective: The aim: To determine malignant transformation and progression ability of high grade and low grade prostate intraepithelial neoplasia with the help of immunohistochemical method., Patients and Methods: Materials and methods: The results of examination of 93 patients with PIN (50 patients with high grade PIN and 43 patients with low grade PIN) were assessed comparatively using immunohistochemical markers. Semiquantitative method was used to evaluate !"-67, #63 and AMACR tissue expression with four grades from "+" to "++++" or from 1 to 4 points: '+' - low reaction, '++' - poor reaction, '+++' - moderate reaction and '++++" - intense reaction., Results: Results: There were statistically significant differences in immunohistochemical expression rates between HGPIN and LGPIN. Patients with HGPIN had higher Ki-67 and AMACR expression rate and lower p63 expression rate than patients with LGPIN. Intense and moderate Ki-67 expression was detected in HGPIN more often, in 24 % and 11 % respectively. Low and moderate AMACR expression was determined in HGPIN more often, in 28 % and 5 % respectively. Low and not evident p63 expression was observed in HGPIN more often, in 36 % and 8 % respectively., Conclusion: Conclusions: HGPIN has common morphological peculiarities with prostate adenocarcinoma. Immunohistochemical detection of Ki-67, p63 and AMACR is aimed to differentiate among patients with PIN a group of high malignant transformation risk.

In Brief: Maternal obesity plus high-fat diet in breastfeeding induces stromal hyperplasia and diffuse acinar atrophy in the rat prostate at aging, related to dyslipidemia and testosterone reduction. The high-lipid nutritional environment from intrauterine and throughout life favors the development of prostatic intraepithelial neoplasia and aggravated degenerative alterations in the gland., Abstract: Maternal obesity and high-fat diet (HFD) affect permanently prostate histophysiology in adulthood, but the consequences during aging are unknown. Here, we evaluated the prostate alterations in middle-aged rats subjected to a high-lipid nutritional environment (HLE) in different ontogenetic periods. Wistar rats (56 weeks of age) were assigned into groups exposed to standard nutrition (C) or HLE during gestation (G), gestation and lactation (GL), from lactation onward (L), from weaning onward (W) and from gestation onward (AL). HLE in the periods after weaning consisted of HFD (20% fat), and during gestation and lactation it also included previous maternal obesity induced by the HFD. HLE increased total cholesterol and triglyceride levels in all groups and led to insulin resistance in GL and AL and obesity in L. Serum testosterone levels decreased ~67% in GL, ~146% in L and W, and ~233% in AL. Histological and stereological analysis revealed an increment of the stromal compartment and collagen fibers in the prostates of all HLE groups, as well as degenerative lesions, such as cell vacuolation and prostate concretions. HLE aggravated acinar atrophy in G, GL, and L, and in AL it reached more than 50% of the prostate area for most animals. The foci of prostatic intraepithelial neoplasia increased in AL. Tissue expression of androgen receptor did not vary among groups, except for a higher stromal expression for G and GL. Even when restricted to gestation and lactation, HLE induces diffuse acinar atrophy in the aging prostate and worsens degenerative and premalignant lesions when it continues throughout life.

Introduction and Objectives: Prostate cancer is one of the most commonly diagnosed cancers in American men. Apelin is an endogenous peptide identified as the ligand of the G protein-associated apelin receptor. Apelin and apelin receptor have many tissues distribution and they participate in pathological processes, such as cancer. Apelin stimulates cancer angiogenesis. However, there are insufficient data in the literature regarding the role of apelin/apelin receptor in normal tissue, highgrade prostatic intraepithelial neoplasia, and prostatic adenocarcinoma tissues. Therefore, this study aimed to investigate the apelin and apelin receptor expression levels in tissues of normal prostate tissue, high-grade prostatic intraepithelial neoplasia, and prostatic adenocarcinoma., Materials and Methods: In this study, 38 samples of patients undergoing radical prostatectomy were used. Among 38 samples; 20 patients were with prostatic adenocarcinoma, 18 patients were with high-grade prostatic intraepithelial neoplasia and adjacent normal prostatic tissue areas. The immunolocalisation of apelin and apelin receptor in these tissues were determined immunohistochemically., Results: Apelin and apelin receptor expressions were higher in prostatic adenocarcinoma than normal prostate tissue and high-grade prostatic intraepithelial neoplasia. Apelin receptor expression was also increased in high-grade prostatic intraepithelial neoplasia compared to normal tissue., Conclusion: Apelin and apelin receptor are increase in the process of prostate carcinogenesis. This increase may adversely affect the clinical course of prostate cancer patients by stimulating angiogenesis, which is important for invasion and metastasis in prostate cancer.

Substantial data posit estrogen receptors (ERs) as promising targets for prostate cancer (PCa) therapeutics. However, the trials on assessing the chemo-preventive or therapeutic potential of ER targeting drugs or selective estrogen receptor modulators (SERMs) have not yet established their clinical benefits. This could be ascribed to a possible modulation in the ER expression during PCa progression. Further it is warranted to test various ER targeting drugs in appropriate preclinical models that simulate human ER expression pattern during PCa progression. The study was undertaken to revisit the existing data on the epithelial ER expression pattern in human cancerous prostates and experimentally determine whether these patterns are replicated in TRAMP (Transgenic Adenocarcinoma of Mouse Prostate) mice, a model for human PCa. Estradiol (E2) binding to the plasma membrane of the epithelial cells and its modulation during the PCa progression in TRAMP were also investigated. A reassessment of the existing data revealed a trend towards downregulation in the epithelial expression of wild-type ESR1 transcripts in high-grade PCa, compared to non-cancerous prostate in humans. Next, epithelial cell-enriched populations from TRAMP prostates (TP) displaying low-grade prostatic intraepithelial neoplasia (LGPIN), high-grade PIN (HGPIN), HGPIN with well-differentiated carcinoma (PIN + WDC), WDC (equivalent to grade 2/3 human PCa), and poorly-differentiated carcinoma (PDC-equivalent to grade 4/5 human PCa) revealed significantly higher Esr1 and Esr2 levels in HGPIN and significantly reduced levels in WDC, compared to respective age-matched control prostates. These patterns for the nuclear ERs were similar to the trend shown by E2 binding to the plasma membrane of the epithelial cells during PCa progression in TRAMP. E2 binding to epithelial cells (EpCAM+), though significantly higher in TPs displaying LGPIN, decreased significantly as the disease progressed to WDC. The study highlights a reduction in the epithelial ESR level with the PCa progression and this pattern was evident in both humans and TRAMP. These observations may have major implications in refining PCa therapeutics targeting ER., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Prostate cancer (PCa) is a leading cause of cancer-related deaths. The slow evolution of precancerous lesions to malignant tumors provides a broad time frame for preventing PCa. To characterize prostatic intraepithelial neoplasia (PIN) progression, we conducted longitudinal studies on Pten (i)pe-/- mice that recapitulate prostate carcinogenesis in humans. We found that early PINs are hypoxic and that hypoxia-inducible factor 1 alpha (HIF1A) signaling is activated in luminal cells, thus enhancing malignant progression. Luminal HIF1A dampens immune surveillance and drives luminal plasticity, leading to the emergence of cells that overexpress Transglutaminase 2 (TGM2) and have impaired androgen signaling. Elevated TGM2 levels in patients with PCa are associated with shortened progression-free survival after prostatectomy. Last, we show that pharmacologically inhibiting HIF1A impairs cell proliferation and induces apoptosis in PINs. Therefore, our study demonstrates that HIF1A is a target for PCa prevention and that TGM2 is a promising prognostic biomarker of early relapse after prostatectomy.

Macrophages are increased in human benign prostatic hyperplasia and prostate cancer. We generate a Pb-Csf1 mouse model with prostate-specific overexpression of macrophage colony-stimulating factor (M-Csf/Csf1). Csf1 overexpression promotes immune cell infiltration into the prostate, modulates the macrophage polarity in a lobe-specific manner, and induces senescence and low-grade prostatic intraepithelial neoplasia (PIN). The Pb-Csf1 prostate luminal cells exhibit increased stem cell features and undergo an epithelial-to-mesenchymal transition. Human prostate cancer patients with high CSF-1 expression display similar transcriptional alterations with the Pb-Csf1 model. P53 knockout alleviates senescence but fails to progress PIN lesions. Ablating epithelial Gp130 but not Il1r1 substantially blocks PIN lesion formation. The androgen receptor (AR) is downregulated in Pb-Csf1 mice. ChIP-Seq analysis reveals altered AR binding in 2482 genes although there is no significant widespread change in global AR transcriptional activity. Collectively, our study demonstrates that increased macrophage infiltration causes PIN formation but fails to transform prostate cells., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Epidemiological data have linked vitamin D deficiency to the onset and severity of various cancers, including prostate cancer, and although in vitro studies have demonstrated anticancer activities for vitamin D, clinical trials provided conflicting results. To determine the impact of vitamin D signaling on prostatic precancerous lesions, we treated genetically engineered Pten (i)pe-/- mice harboring prostatic intraepithelial neoplasia (PIN) with Gemini-72, a vitamin D analog with reported anticancer activities. We show that this analog induces apoptosis in senescent PINs, normalizes extracellular matrix remodeling by stromal fibroblasts, and reduces the prostatic infiltration of immunosuppressive myeloid-derived suppressor cells. Moreover, single-cell RNA-sequencing analysis demonstrates that while a subset of luminal cells expressing Krt8, Krt4, and Tacstd2 (termed luminal-C cells) is lost by such a treatment, antiapoptotic pathways are induced in persistent luminal-C cells. Therefore, our findings delineate the distinct responses of PINs and the microenvironment to Gemini-72, and shed light on mechanisms that limit treatment's efficacy., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).)

Diagnosis of Prostatic adenocarcinoma (PAC) is still a problematic issue. The objective of this study was to evaluate the diagnostic and prognostic value of ERG immunohistochemical (IHC) expression compared to MAGI2., Materials and Methods: This study was conducted on 56 cases of PAC and 29 cases of nodular prostatic hyperplasia (NPH). IHC staining for ERG and MAGI2 was applied to archival formalin-fixed paraffin-embedded blocks. Semi-quantitative scoring was compared and correlated with clinicopathologic parameters and the Ki-67 index., Results: Revealed positive ERG in 51.8% of PAC while all NPH cases were negative. On the other hand, MAGI2 was detected in 91.1% of PAC versus 17.2% of NPH. Using ROC curve, the ERG showed 53.6% sensitivity, 100% specificity, 76.5% diagnostic accuracy (DA) and area under the ROC curve 0.768 in comparison to MAGI2 that showed (91.1%, 86.2%, 88.25% and 0.948 respectively). Analysis of the combined use of the two markers revealed 95% sensitivity, 100% specificity, and 94% DA when tested synchronously. Moreover, a statistically significant inverse relationship could be detected between ERG expression and the Gleason grading group (P = 0.01) and Ki-67 index (P < 0.001). In addition, high-grade prostatic intraepithelial neoplasia (HGPIN) adjacent to carcinoma; showed positive expressions in (1/11 cases, 9.11%) for ERG and (6/11 cases, 54%) for MAGI2., Conclusion: This study recommends using both ERG and MAGI2 in a cocktail for better diagnostic validity of PAC. Only ERG expression could be a good prognostic indicator., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The risk factors for prostate cancer include a high-fat diet and obesity, both of which are associated with an altered cell environment including increased inflammation. It has been shown that chronic inflammation due to a high-fat diet or bacterial infection has the potential to accelerate prostate cancer as well as its precursor, prostatic intraepithelial neoplasia (PIN), development. However, the underlying mechanism of how chronic inflammation promotes prostate cancer development, especially PIN, remains unclear. In this study, we showed that more macrophages were present in PIN areas as compared to the normal areas of human prostate. When co-culturing PIN cells with macrophages in 3D, more PIN cells had nuclear localized cyclin D1, indicating that macrophages enhanced PIN cell proliferation. We identified ICAM-1 and CCL2 as chemoattractants expressed by PIN cells to recruit macrophages. Furthermore, we discovered that macrophage-secreted cytokines including C5a, CXCL1, and CCL2 were responsible for increased PIN cell proliferation. These three cytokines activated ERK and JNK signaling in PIN cells through a ligand-receptor interaction. However, only blockade of ERK abolished macrophage cytokines-induced cell proliferation of PIN. Overall, our results provide a mechanistic view on how macrophages activated through chronic inflammation can expedite PIN progression during prostate cancer development. The information from our work can facilitate a comprehensive understanding of prostate cancer development, which is required for improvement of current strategies for prostate cancer therapy., (© 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Chemoprevention trials for prostate cancer by androgen receptor or androgen synthesis inhibition have proven ineffective. Recently, it has been demonstrated that the histone methlytransferase, EZH2 is deregulated in mouse and human high-grade prostatic intraepithelial neoplasia (HG-PIN). Using preclinical mouse and human models of prostate cancer, we demonstrate that genetic and chemical disruption of EZH2 expression and catalytic activity reversed the HG-PIN phenotype. Furthermore, inhibition of EZH2 function was associated with loss of cellular proliferation and induction of Tp53-dependent senescence. Together, these data provide provocative evidence for EZH2 as an actionable therapeutic target toward prevention of prostate cancer., (©2020 American Association for Cancer Research.)

Since the industrial revolution, all living beings have become susceptible to numerous sources of aluminum (Al) exposure. In addition to causing proven toxicity in many organs and systems, Al can also have estrogenic activity when absorbed by the body. The reproductive organs are commonly affected by environmental pollutants with estrogenic activity, but little is known about the effects of Al on the prostate and gonads. Therefore, the aim of this study was to evaluate the effects of subchronic Al exposure on the prostate and gonads of male and female adult gerbils. After 30 days of oral exposure to aluminum chloride (10 mg/kg/day), the animals were euthanized and the organs processed for cytochemical, ultrastructural, and biochemical assays. Ventral male prostates exposed to Al became hyperplastic and showed signs of cell aging. In addition, the male prostate showed decreased catalase (CAT) and superoxide dismutase (SOD) activity. The female prostate was structurally more affected than the ventral male prostate, since it presented hyperplasia and punctual foci of inflammation and prostatic intraepithelial neoplasia. However, CAT and SOD activities did not change in this gland. In the testis, Al promoted immature germ cell detachment and degeneration, as well as reduced CAT activity. In the ovaries, Al caused reduction in folliculogenesis and decreased SOD activity. Together, these results indicate that Al is toxic to the prostate and gonads of adult gerbils and that continuous exposure to this metal can impair the fertility of individuals of both sexes., Competing Interests: Declaration of Competing Interest The authors declare that there are no conflicts of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Galectin-3 (Gal-3) is an extracellular matrix glycan-binding protein with several immunosuppressive and pro-tumor functions. The role of Galectin-3 in cancer stem-like cells (CSCs) is poorly investigated. Here, we show that prostate CSCs also colonizing prostate-draining lymph nodes of transgenic adenocarcinoma of the mouse prostate (TRAMP) mice overexpress Gal-3. Gal-3 contributes to prostate CSC-mediated immune suppression because either Gal-3 silencing in CSCs, or co-culture of CSCs and T cells in the presence of the Gal-3 inhibitor N-Acetyl-D-lactosamine rescued T cell proliferation. N-Acetyl-D-lactosamine also rescued the proliferation of T cells in prostate-draining lymph nodes of TRAMP mice affected by prostate intraepithelial neoplasia. Additionally, Gal-3 impacted prostate CSC tumorigenic and metastatic potential in vivo , as Gal-3 silencing in prostate CSCs reduced both primary tumor growth and secondary invasion. Gal-3 was also found expressed in more differentiated prostate cancer cells, but with different intracellular distribution as compared to CSCs, which suggests different functions of Gal-3 in the two cell populations. In fact, the prevalent nuclear and cytoplasmic distribution of Gal-3 in prostate CSCs made them less susceptible to apoptosis, when compared to more differentiated prostate cancer cells, in which Gal-3 was predominantly intra-cytoplasmic. Finally, we found Gal-3 expressed in human and mouse prostate intraepithelial neoplasia lesions and in metastatic lymph nodes. All together, these findings identify Gal-3 as a key molecule and a potential therapeutic target already in the early phases of prostate cancer progression and metastasis., (Copyright © 2020 Caputo, Grioni, Brambillasca, Monno, Brevi, Freschi, Piras, Elia, Pieri, Baccega, Lombardo, Galli, Briganti, Doglioni, Jachetti and Bellone.)

Background: Glutathione peroxidase 3 (Gpx3) protects cells from oxidative stress, and its reduced expression in human prostate cancer has been reported., Objectives: We hypothesized that Gpx3 might play an important role in the development of prostatic intraepithelial neoplasia (PIN), a pre-cancerous state of the prostate, and aimed to highlight the underlying molecular mechanism., Materials and Methods: The following double-knockout mice Nkx3.1-/-; Gpx3+/+, Nkx3.1-/-; Gpx3+/-, Nkx3.1-/-; Gpx3-/- were produced. Randomly divided animals were weighed, and their genitourinary tract (GUT) weights were determined after euthanasia at 4, 8, and 12 months. The mRNA expression of the genes involved in oxidative stress and Wnt signaling was analyzed in the prostate. Histopathology, ROS, and superoxide dismutase (SOD) activities were also measured., Results: Loss of Gpx3 did not affect body weight and GUT weight in Nkx3.1 knockout mice. The mRNA expression of SOD3, iNOS, Hmox, and CISD2, which are associated with oxidative stress, was increased in Nkx3.1-/-; Gpx3-/- mice at 4 months but decreased at 8 and 12 months. There was no change in β-catenin and its targets associated with Wnt signaling. Increased ROS and decreased SOD activity were observed in Nkx3.1-/-; Gpx3-/- mice at 12 months of age. The histopathologic score and epithelium thickness were increased, and lumen area was decreased in Gpx3 knockout mice., Discussion and Conclusions: Gpx3 loss increased the hyperplasia of PIN in the pre-cancerous stage of the prostate. Loss of Gpx3 induced oxidative stress. Histopathologically, no invasive carcinoma was identified, and Gpx3 loss did not increase Wnt/β-catenin signaling. Further research on the role of GPX3 in the transition of PIN to invasive carcinoma is needed. We show, for the first time, that the antioxidant enzyme GPX3 plays a vital role in inhibiting hyperplasia in the PIN stage of the prostate gland in vivo., (© 2020 American Society of Andrology and European Academy of Andrology.)

Background: ETV4 is one of the ETS proteins overexpressed in prostate cancer (PC) as a result of recurrent chromosomal translocations. In human prostate cell lines, ETV4 promotes migration, invasion, and proliferation; however, its role in PC has been unclear. In this study, we have explored the effects of ETV4 expression in the prostate in a novel transgenic mouse model., Methods: We have created a mouse model with prostate-specific expression of ETV4 (ETV4 mice). By histochemical and molecular analysis, we have investigated in these engineered mice the expression of p21, p27, and p53. The implications of our in vivo findings have been further investigated in human cells lines by chromatin-immunoprecipitation (ChIP) and luciferase assays., Results: ETV4 mice, from two independent transgenic lines, have increased cell proliferation in their prostate and two-thirds of them, by the age of 10 months, developed mouse prostatic intraepithelial neoplasia (mPIN). In these mice, cdkn1a and its p21 protein product were reduced compared to controls; p27 protein was also reduced. By ChIP assay in human prostate cell lines, we show that ETV4 binds to a specific site (-704/-696 bp upstream of the transcription start) in the CDKN1A promoter that was proven, by luciferase assay, to be functionally competent. ETV4 further controls CDKN1A expression by downregulating p53 protein: this reduction of p53 was confirmed in vivo in ETV4 mice., Conclusions: ETV4 overexpression results in the development of mPIN but not in progression to cancer. ETV4 increases prostate cell proliferation through multiple mechanisms, including downregulation of CDKN1A and its p21 protein product: this in turn is mediated through direct binding of ETV4 to the CDKN1A promoter and through the ETV4-mediated decrease of p53. This multi-faceted role of ETV4 in prostate cancer makes it a potential target for novel therapeutic approaches that could be explored in this ETV4 transgenic model.

Introduction: CIP2A is an oncoprotein involved in the progression of several human malignancies. It has recently been described as a prognostic marker in many cancers. The present study aimed to investigate the immunohistochemical expression of CIP2A in benign prostatic hyperplasia (BPH), high grade prostatic intraepithelial neoplasia (HGPIN) and prostate cancer (PC), and to analyse the association with the clinicopathological parameters in PC cases to define its role in the development and progression of PC., Materials and Methods: Immunohistochemical staining for CIP2A was performed on the tissue microarray sections of 105 PC, 27 HGPIN and 27 BPH tissues. The CIP2A expression scores were compared with several clinicopathological parameters., Results: CIP2A was expressed in 96,2% of PC, 55,6% of HGPIN and 40,7% of BPH tissues. The expression of CIP2A in PC was significantly higher than in HGPIN (p<0.0001) and BPH (p<0.0001) cases. CIP2A expression score was significantly associated with Gleason score (p=0.032) and lymphovascular invasion (p=0.039). Nevertheless, there was no statistically significant association between the expression of CIP2A and perineural invasion, pT stage, metastasis and recurrence (p>0.05). Multivariate analysis indicated that GS, lymphovascular invasion, distant metastasis were independent prognostic factors for PC patients but, CIP2A expression score was not found to be a prognostic factor. Additionally, there was no significant difference between the survival times of patients according to CIP2A expression (p=0.174)., Conclusions: According to our results, the expression of CIP2A protein is increased in PC and its expression may be involved in the development, differentiation, and aggressiveness of PC. However, further studies are needed to confirm our findings and to clarify the role of CIP2A in the development of PC.

Introduction: The metabolic alterations reflecting the influence of prostate cancer cells can be captured through metabolomic profiling., Objective: To characterize the plasma metabolomic profile in prostatic intraepithelial neoplasia (PIN) and prostate cancer (PCa)., Methods: Metabolomics analyses were performed in plasma samples from individuals classified as non-cancerous control (n = 36), with PIN (n = 16), or PCa (n = 27). Untargeted [26 moieties identified after pre-processing by gas chromatography/mass spectrometry (GC/MS)] and targeted [46 amino acids, carbohydrates, organic acids and fatty acids by GC/MS, and 16 nucleosides and amino acids by ultra performance liquid chromatography-triple quadrupole/mass spectrometry (UPLC-TQ/MS)] analyses were performed. Prostate specific antigen (PSA) concentrations were measured in all samples. In PCa patients, the Gleason scores were determined., Results: The metabolites that were best discriminated (p < 0.05, FDR < 0.2) for the Kruskal-Wallis test with Dunn's post-hoc comparing the control versus the PIN and PCa groups included isoleucine, serine, threonine, cysteine, sarcosine, glyceric acid, among several others. PIN was mainly characterized by alterations on steroidogenesis, glycine and serine metabolism, methionine metabolism and arachidonic acid metabolism, among others. In the case of PCa, the most predominant metabolic alterations were ubiquinone biosynthesis, catecholamine biosynthesis, thyroid hormone synthesis, porphyrin and purine metabolism. In addition, we identified metabolites that were correlated to the PSA [i.e. hypoxanthine (r = - 0.60, p < 0.05; r = - 0.54, p < 0.01) and uridine (r = - 0.58, p < 0.05; r = - 0.50, p < 0.01) in PIN and PCa groups, respectively] and metabolites that were significantly different in PCa patients with Gleason score < 7 and ≥ 7 [i.e. arachidonic acid, median (P25-P75) = 883.0 (619.8-956.4) versus 570.8 (505.6-651.8), respectively (p < 0.01)]., Conclusions: This human plasma metabolomic assessment contributes to the understanding of the unique metabolic features exhibited in PIN and PCa and provides a list of metabolites that can have the potential to be used as biomarkers for early detection of disease progression and management.

Background: Corpora amylacea (CAM), in benign prostatic acini, contain acute-phase proteins. Do CAM coincide with carcinoma?, Methods: Within 270 biopsies, 83 prostatectomies, and 33 transurethral resections (TURs), CAM absence was designated CAM 0; corpora in less than 5% of benign acini: CAM 1; in 5% to 25%: CAM 2; in more than 25%: CAM 3. CAM were compared against carcinoma presence, clinicopathologic findings, and grade groups (GG) 1 to 2 vs 3 to 5. The frequency of CAM according to anatomic zone was counted. A pilot study was conducted using paired initial benign and repeat biopsies (33 benign, 24 carcinoma)., Results: A total of 68.9% of biopsies, 96.4% of prostatectomies, and 66.7% of TURs disclosed CAM. CAM ≥1 was common at an older age (P = .019). In biopsies, 204 cases (75%) had carcinoma; and CAM of 2 to 3 (compared to 0-1) were recorded in 25.0% of carcinomas but only 7.4% of benign biopsies (P = .005; odds ratio [OR] = 5.1). CAM correlated with high percent Gleason pattern 3, low GG (P = .035), and chronic inflammation (CI). CI correlated inversely with carcinoma (P = .003). CAM disclosed no association with race, body mass index, serum prostate specific antigen (PSA), acute inflammation (in biopsies), atrophy, or carcinoma volume. With CAM 1, the odds of GG 3 to 5 carcinoma, by comparison to CAM 0, decreased more than 2× (OR = 0.48; P = .032), with CAM 2, more than 3× (OR = 0.33; P = .005), and with CAM 3, almost 3× (OR = 0.39, P = .086). For men aged less than 65, carcinoma predictive model was: Score = (2 × age) + (5 × PSA) - (20 × degree of CAM); using our data, area under the ROC curve was 78.17%. When the transition zone was involved by cancer, it showed more CAM than in cases where it was uninvolved (P = .012); otherwise zonal distributions were similar. In the pilot study, CAM ≥1 predicted carcinoma on repeat biopsy (P < .05; OR = 8), as did CAM 2 to 3 (P < .0001; OR = 30). CI was not significant, and CAM retained significance after adjusting for CI., Conclusion: CAM correlate with carcinoma. Whether abundant CAM in benign biopsies adds value amidst high clinical suspicion, warrants further study., (© 2020 Wiley Periodicals, Inc.)

We genetically engineered expression of an activated form of P110 alpha, the catalytic subunit of PI3K, in mouse prostate epithelium to create a mouse model of direct PI3K activation (Pbsn-cre4Prb;PI3K GOF/+ ). We hypothesized that direct activation would cause rapid neoplasia and cancer progression. Pbsn-cre4Prb;PI3K GOF/+ mice developed widespread prostate intraepithelial hyperplasia, but stromal invasion was limited and overall progression was slower than anticipated. However, the model produced profound and progressive stromal remodeling prior to explicit epithelial neoplasia. Increased stromal cellularity and inflammatory infiltrate were evident as early as 4 months of age and progressively increased through 12 months of age, the terminal endpoint of this study. Prostatic collagen density and phosphorylated SMAD2-positive prostatic stromal cells were expansive and accumulated with age, consistent with pro-fibrotic TGF-β pathway activation. Few reported mouse models accumulate prostate-specific collagen to the degree observed in Pbsn-cre4Prb;PI3K GOF/+ . Our results indicate a signaling process beginning with prostatic epithelial PI3K and TGF-β signaling that drives prostatic stromal hypertrophy and collagen accumulation. These mice afford a unique opportunity to explore molecular mechanisms of prostatic collagen accumulation that is relevant to cancer progression, metastasis, inflammation and urinary dysfunction. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd., (© 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

RalGTPase-activating protein (RalGAP) is an important negative regulator of small GTPases RalA/B that mediates various oncogenic signaling pathways in various cancers. Although the Ral pathway has been implicated in prostate cancer (PCa) development and progression, the significance of RalGAP in PCa has been largely unknown. We examined RalGAPα2 expression using immunohistochemistry on two independent tissue microarray sets. Both datasets demonstrated that the expression of RalGAPα2 was significantly downregulated in PCa tissues compared to adjacent benign prostatic epithelia. Silencing of RalGAPα2 by short hairpin RNA enhanced migration and invasion abilities of benign and malignant prostate epithelial cell lines without affecting cell proliferation. Exogenous expression of wild-type RalGAP, but not the GTPase-activating protein activity-deficient mutant of RalGAP, suppressed migration and invasion of multiple PCa cell lines and was phenocopied by pharmacological inhibition of RalA/B. Loss of Ralgapa2 promoted local microscopic invasion of prostatic intraepithelial neoplasia without affecting tumor growth in a Pten-deficient mouse model for prostate tumorigenesis. Our findings demonstrate the functional significance of RalGAP downregulation to promote invasion ability, which is a property necessary for prostate carcinogenesis. Thus, loss of RalGAP function has a distinct role in promoting progression from prostatic intraepithelial neoplasia to invasive adenocarcinoma., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

ELL-associated factor 1 is a transcription elongation factor that shares significant homology and functional similarity to the androgen-responsive prostate tumor suppressor ELL-associated factor 2. EAF2 is frequently down-regulated in advanced prostate cancer and Eaf2 deletion in the mouse induced the development of murine prostatic intraepithelial neoplasia. Here we show that similar to EAF2, EAF1 is frequently down-regulated in advanced prostate cancer. Co-downregulation of EAF1 and EAF2 occurred in 40% of clinical specimens with Gleason score >7. We developed and characterized a murine model of prostate-epithelial specific deletion of Eaf1 in the prostate and crossed it with our previously generated mouse with conventional deletion of Eaf2. The prostates of Eaf1 deletion mice displayed murine prostatic intraepithelial neoplasia lesions with increased proliferation and inflammation. Combined deletion of Eaf1 and Eaf2 in the murine model induced an increased incidence in mPIN lesions characterized by increased proliferation and CD3+ T cells and CD19+ B cells infiltration compared to individual deletion of either Eaf1 or Eaf2 in the murine prostate. These results suggest that EAF1 may play a tumor suppressive role in the prostate. Cooperation between EAF1 and EAF2 may be important for prostate maintaining prostate epithelial homeostasis, and concurrent loss of these two tumor suppressors may promote prostate tumorigenesis and progression., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

The ETS transcription factor ETV1 is frequently overexpressed in aggressive prostate cancer, which is one underlying cause of this disease. Accordingly, transgenic mice that prostate-specifically overexpress ETV1 develop prostatic intraepithelial neoplasia. However, progression to the adenocarcinoma stage is stifled in these mice, suggesting that inhibitory pathways possibly preclude ETV1 from exerting its full oncogenic potential. Here we provide evidence that TGF-β/SMAD signaling represents such an inhibitory pathway. First, we discovered that ETV1 forms complexes with SMAD4. Second, SMAD2, SMAD3 and SMAD4 overexpression impaired ETV1's ability to stimulate gene transcription. Third, TGF-β1 inhibited ETV1-induced invasion by benign RWPE-1 prostate cells. Fourth, increased expression of SMAD3 and SMAD4 was observable in prostates of ETV1 transgenic mice. Conversely, we found that ETV1 may enhance TGF-β signaling in PC3 prostate cancer cells, revealing a different facet of the ETV1/TGF-β interplay. Altogether, these data provide more insights into the regulation and action of ETV1 and additionally suggest that TGF-β/SMAD signaling exerts its tumor suppressive activity, at least in part, by curtailing the oncogenic potential of ETV1 in prostatic lesions.

In vitro experiments demonstrated that stimulation of Toll-like receptor 9 (TLR-9) by synthetic TLR-9 ligands induces the invasion of TLR-9-expressing prostate cancer cells through matrix metalloproteinase 13 (MMP-13). However, the clinical value of TLR-9 and MMP-13 co-expression in the pathophysiology of the prostate is unknown. In the study, we evaluated the expression levels and clinical significance of the TLR-9 and MMP-13 in a series of prostate tissues. One hundred and eighty prostate tissues including prostate cancer (PCa) (n = 137), high-grade prostatic intraepithelial neoplasia (HPIN) (n = 18) and benign prostatic hyperplasia (BPH) (n = 25) were immunostained for the TLR-9 and MMP-13 markers. Subsequently, the correlation between the TLR-9 and MMP-13 staining scores and clinicopathological parameters was obtained. Higher expressions of TLR-9 and MMP-13 were found in PCa and high-grade prostatic intraepithelial neoplasia compared to benign prostatic hyperplasia tissues. Among PCa samples, a positive relationship was revealed between the MMP-13 expression and Gleason score (P < 0.001). There was a significant correlation between TLR-9 expression and regional lymph node involvement (P = 0.04). The expression patterns of TLR-9 and MMP-13 markers demonstrated a reciprocal significant correlation between the two markers in the same series of prostate samples (P < 0.001). Furthermore, the Gleason score of TLR-9 high /MMP-13 high and TLR-9 low /MMP-13 low phenotypes showed a significant difference (P = 0.002). Higher expressions of TLR-9 and MMP-13 can confer aggressive behaviour to PCa. Therefore, these markers may be used as a valuable target for tailored therapy of PCa., (© 2019 The Authors. International Journal of Experimental Pathology © 2019 International Journal of Experimental Pathology.)

The tumor suppressor p16Ink4a, encoded by the INK4a gene, is an inhibitor of cyclin D-dependent kinases 4 and 6, CDK4 and CDK6. This inhibition prevents the phosphorylation of the retinoblastoma protein (pRb), resulting in cellular senescence through inhibition of E2F-mediated transcription of S phase genes required for cell proliferation. The p16Ink4a plays an important role in tumor suppression, whereby its deletion, mutation, or epigenetic silencing is a frequently observed genetic alteration in prostate cancer. To assess its roles and related molecular mechanisms in prostate cancer initiation and progression, we generated a mouse model with conditional deletion of p16Ink4a in prostatic luminal epithelium. The mice underwent oncogenic transformation and developed prostatic intraepithelial neoplasia (PIN) from eight months of age, but failed to develop prostatic tumors. Given the prevalence of aberrant androgen signaling pathways in prostate cancer initiation and progression, we then generated R26hARL/wt:p16L/L: PB-Cre4 compound mice, in which conditional expression of the human AR transgene and deletion of p16Ink4a co-occur in prostatic luminal epithelial cells. While R26hARL/wt:PB-Cre4 mice showed no visible pathological changes, R26hARL/wt:p16L/L: PB-Cre4 compound mice displayed an early onset of high-grade PIN (HGPIN), prostatic carcinoma, and metastatic lesions. Strikingly, we observed tumors resembling human sarcomatoid carcinoma with intermixed focal regions of signet ring cell carcinoma (SRCC) in the prostates of the compound mice. Further characterization of these tumors showed they were of luminal epithelial cell origin, and featured characteristics of epithelial to mesenchymal transition (EMT) with enhanced proliferative and invasive capabilities. Our results not only implicate a biological role for AR expression and p16Ink4a deletion in the pathogenesis of prostatic SRCC, but also provide a new and unique genetically engineered mouse (GEM) model for investigating the molecular mechanisms for SRCC development., Competing Interests: The authors have declared that no competing interests exist.

Increased de novo synthesis of fatty acids is a rather unique and targetable mechanism of human prostate cancer. We have shown previously that oral administration of sulforaphane (SFN) significantly inhibits the incidence and/or burden of prostatic intraepithelial neoplasia and well-differentiated adenocarcinoma in TRansgenic Adenocarcinoma of Mouse Prostate (TRAMP) mice. The present study used cellular models of prostate cancer and archived plasma/adenocarcinoma tissues and sections from the TRAMP study to demonstrate inhibition of fatty acid synthesis by SFN treatment in vitro and in vivo. Treatment of androgen-responsive (LNCaP) and castration-resistant (22Rv1) human prostate cancer cells with SFN (5 and 10 μM) resulted in downregulation of protein and mRNA levels of acetyl-CoA carboxylase 1 (ACC1) and fatty acid synthase (FASN), but not ATP citrate lyase. Protein and mRNA levels of carnitine palmitoyltransferase 1A (CPT1A), which facilitates fatty acid uptake by mitochondria for β-oxidation, were also decreased following SFN treatment in both cell lines. Immunohistochemistry revealed a significant decrease in expression of FASN and ACC1 proteins in prostate adenocarcinoma sections of SFN-treated TRAMP mice when compared with controls. SFN administration to TRAMP mice resulted in a significant decrease in plasma and/or prostate adenocarcinoma levels of total free fatty acids, total phospholipids, acetyl-CoA and ATP. Consistent with these results, number of neutral lipid droplets was lower in the prostate adenocarcinoma sections of SFN-treated TRAMP mice than in control tumors. Collectively, these observations indicate that prostate cancer chemoprevention by SFN in TRAMP mice is associated with inhibition of fatty acid metabolism.

Background: The tumor promoting or counteracting effects of the immune response to cancer development are thought to be mediated to some extent by the infiltration of regulatory T cells (T regs ). In the present study we evaluated the prevalence of T reg populations in stromal and epithelial compartments of normal, post atrophic hyperplasia (PAH), prostatic intraepithelial neoplasia (PIN), and tumor lesions in men with and without prostate cancer., Methods: Study subjects were 102 men consecutively diagnosed with localized prostate cancer undergoing radical prostatectomy and 38 men diagnosed with bladder cancer undergoing cystoprostatectomy without prostate cancer at the pathological examination. Whole mount sections from all patients were evaluated for the epithelial and stromal expression of CD4 + T regs and CD8 + T regs in normal, PAH, PIN, and tumor lesions. A Friedmańs test was used to investigate differences in the mean number of T regs across histological lesions. Logistic regression was used to estimate crude and adjusted odds ratios (OR) for prostate cancer for each histological area., Results: In men with prostate cancer, similarly high numbers of stromal CD4 + T regs were identified in PAH and tumor, but CD4 + T regs were less common in PIN. Greater numbers of epithelial CD4+ T regs in normal prostatic tissue were positively associated with both Gleason score and pT-stage. We observed a fourfold increased risk of prostate cancer in men with epithelial CD4 + T regs in the normal prostatic tissue counterpart., Conclusions: Our results may suggest a possible pathway through which PAH develops directly into prostate cancer in the presence of CD4 + T regs and indicate that transformation of the anti-tumor immune response may be initiated even before the primary tumor is established., (© 2017 The Authors. The Prostate Published by Wiley Periodicals 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.)

The E3 ubiquitin ligase adaptor speckle-type POZ protein (SPOP) is frequently dysregulated in prostate adenocarcinoma (PC), via either somatic mutations or mRNA downregulation, suggesting an important tumour suppressor function. To examine its physiologic role in the prostate epithelium in vivo, we generated mice with prostate-specific biallelic ablation of Spop. These mice exhibited increased prostate mass, prostate epithelial cell proliferation, and expression of c-MYC protein compared to littermate controls, and eventually developed prostatic intraepithelial neoplasia (PIN). We found that SPOP WT can physically interact with c-MYC protein and, upon exogenous expression in vitro, can promote c-MYC ubiquitination and degradation. This effect was attenuated in PC cells by introducing PC-associated SPOP mutants or upon knockdown of SPOP via short-hairpin-RNA, suggesting that SPOP inactivation directly increases c-MYC protein levels. Gene Set Enrichment Analysis revealed enrichment of Myc-induced genes in transcriptomic signatures associated with SPOP MT . Likewise, we observed strong inverse correlation between c-MYC activity and SPOP mRNA levels in two independent PC patient cohorts. The core SPOP MT ;MYC High transcriptomic response, defined by the overlap between the SPOP MT and c-MYC transcriptomic programmes, was also associated with inferior clinical outcome in human PCs. Finally, the organoid-forming capacity of Spop-null murine prostate cells was more sensitive to c-MYC inhibition than that of Spop-WT cells, suggesting that c-MYC upregulation functionally contributes to the proliferative phenotype of Spop knock-out prostates. Taken together, our data highlight SPOP as an important regulator of luminal epithelial cell proliferation and c-MYC expression in prostate physiology, identify c-MYC as a novel bona fide SPOP substrate, and help explain the frequent inactivation of SPOP in human PC. We propose SPOP MT -induced stabilization of c-MYC protein as a novel mechanism that can increase total c-MYC levels in PC cells, in addition to amplification of c-MYC locus.

The aim of the present study was to investigate the immunohistochemical expression of NGF, GDNF and MMP-9 in benign prostatic hyperplasia (BPH), high grade prostatic intraepithelial neoplasia (HGPIN) and prostate cancer (PC), and to analyse their association with the clinicopathological parameters in PC cases. Immunohistochemistry was performed on the tissue microarray (TMA) sections of 30 BPH, 40 HGPIN and 121 primary PC tissues. There was a significant difference regarding the expression of NGF and GDNF between PC and HGPIN (p<0.0001; p<0.0001), and PC and BPH (p=0.001; p<0.0001), but not between HGPIN and BPH (p>0.05). Furthermore MMP-9 expression was significantly different among all groups (PC vs. HGPIN, p<0.0001; PC vs. BPH, p<0.0001; HGPIN vs. BPH, p=0.001). NGF, GDNF and MMP-9 expression was significantly stronger in cases with high Gleason score (p<0.0001, p=0.004, p<0.0001 respectively) and pT stage (p=0.046, p=0.004, p=0.001, respectively) in PC cases. All these markers were also associated with perineural, lymphovascular and extraprostatic invasion (p <0.05). In addition, a positive correlation was found between NGF and MMP-9 (p<0.0001, r=0.435), NGF and GDNF (p<0.0001, r=0.634), and GDNF and MMP-9 (p<0.0001, r=0.670) in PC cases. According to our results we suggest an interaction between NGF, GDNF and MMP-9 during the transition to malignancy in PC. Also this interaction may involve in regulating PC cell differentiation, tumor invasion, progression, and the agressiveness of PC., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

This report develops an analytically validated chromogenic in situ hybridization (CISH) assay using branched DNA signal amplification (RNAscope) for detecting the expression of the 5' external transcribed spacer (ETS) of the 45S ribosomal (r) RNA precursor in formalin-fixed and paraffin-embedded (FFPE) human tissues. 5'ETS/45S CISH was performed on standard clinical specimens and tissue microarrays (TMA) from untreated prostate carcinomas, high-grade prostatic intraepithelial neoplasia (PIN), and matched benign prostatic tissues. Signals were quantified using image analysis software. The 5'ETS rRNA signal was restricted to the nucleolus. The signal was markedly attenuated in cell lines and in prostate tissue slices after pharmacologic inhibition of RNA polymerase I (Pol I) using BMH-21 or actinomycin D, and by RNAi depletion of Pol I, demonstrating validity as a measure of Pol I activity. Clinical human prostate FFPE tissue sections and TMAs showed a marked increase in the signal in the presumptive precursor lesion (high-grade PIN) and invasive adenocarcinoma lesions ( P = 0.0001 and P = 0.0001, respectively) compared with non-neoplastic luminal epithelium. The increase in 5'ETS rRNA signal was present throughout all Gleason scores and pathologic stages at radical prostatectomy, with no marked difference among these. This precursor rRNA assay has potential utility for detection of increased rRNA production in various tumor types and as a novel companion diagnostic for clinical trials involving Pol I inhibition. Implications: Increased rRNA production, a possible therapeutic target for multiple cancers, can be detected with a new, validated assay that also serves as a pharmacodynamic marker for Pol I inhibitors. Mol Cancer Res; 15(5); 577-84. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Basal cells in a simple secretory epithelium adhere to the extracellular matrix (ECM), providing contextual cues for ordered repopulation of the luminal cell layer. Early high-grade prostatic intraepithelial neoplasia (HG-PIN) tissue has enlarged nuclei and nucleoli, luminal layer expansion and genomic instability. Additional HG-PIN markers include loss of α6β4 integrin or its ligand laminin-332, and budding of tumor clusters into laminin-511-rich stroma. We modeled the invasive budding phenotype by reducing expression of α6β4 integrin in spheroids formed from two normal human stable isogenic prostate epithelial cell lines (RWPE-1 and PrEC 11220). These normal cells continuously spun in culture, forming multicellular spheroids containing an outer laminin-332 layer, basal cells (expressing α6β4 integrin, high-molecular-weight cytokeratin and p63, also known as TP63) and luminal cells that secrete PSA (also known as KLK3). Basal cells were optimally positioned relative to the laminin-332 layer as determined by spindle orientation. β4-integrin-defective spheroids contained a discontinuous laminin-332 layer corresponding to regions of abnormal budding. This 3D model can be readily used to study mechanisms that disrupt laminin-332 continuity, for example, defects in the essential adhesion receptor (β4 integrin), laminin-332 or abnormal luminal expansion during HG-PIN progression., (© 2017. Published by The Company of Biologists Ltd.)

Background: Metalloproteinases (MMPs) are relevant modulators of inflammation, tumor microenvironment, cancer invasion and metastasis. They can be regulated by the Low density lipoprotein Receptor-related Protein 1 (LRP-1), a receptor reported to mediate the clearance of lipoproteins, extracellular matrix (ECM) macromolecules and proteinases. The aim of this study was to evaluate the expression of LRP-1, MMP-2 and MMP-9 across various grades of prostatic diseases as benign prostatic hyperplasia (BPH), BPH plus prostatitis (BPH+P), high grade prostatic intraepithelial neoplasia (HGPIN) and prostate cancer (PCa)., Methods: LRP-1 was analyzed using immunohistochemistry and MMPs proteolytic activity by zymography in prostate tissues with different prostatic diseases., Results: LRP-1 was detected in epithelial cells in BPH (16/18), BPH+P (21/21) and HGPIN (6/6), with a staining intensity of 1+, 1+/2+ and 3+, respectively. In PCa, LRP-1 was absent in 19/27 samples while a low expression was observed in 8/27 biopsies. MMP-9 activity was higher and statistically significant in PCa than in BPH (p≤0.01)., Conclusion: Considering that LRP-1, by mediating the clearance of MMPs, is involved in the regulation of ECM remodeling and cell migration, we conclude that a decreased expression of LRP-1 could be involved with the increasing activity of MMPs shown in cancers., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Objectives: We compared the utility of membrane-associated guanylate kinase, WW and PDZ domain-containing protein 2 (MAGI-2) and α-methylacyl CoA (AMACR) by immunohistochemistry in diagnosing prostatic adenocarcinoma., Methods: Seventy-eight radical prostatectomies were used to construct three tissue microarrays with 512 cores, including benign prostatic tissue, benign prostatic hyperplasia, high-grade prostatic intraepithelial neoplasia (HGPIN), and adenocarcinoma. AMACR and MAGI-2 immunohistochemistry were evaluated by visual and image analysis., Results: MAGI-2 and AMACR were significantly higher in adenocarcinoma and HGPIN compared with benign tissue. At H-score cutoffs of 300 and 200, MAGI-2 was more accurate in distinguishing benign from malignant glands than AMACR. Areas under the curve by image and visual analysis were 0.846 and 0.818 for MAGI-2 and 0.937 and 0.924 for AMACR, respectively. The accuracy of MAGI-2 in distinguishing benign from malignant glands on the same core was higher (95% vs 88%)., Conclusions: MAGI-2 could represent a useful adjunct for diagnosis of prostatic adenocarcinoma, especially when AMACR is not discriminatory., (© American Society for Clinical Pathology, 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Steroid sex hormones can induce prostate carcinogenesis, and are thought to contribute to the development of prostate cancer during aging. However, the mechanism for hormone-induced prostate carcinogenesis remains elusive. Here, we report that activating transcription factor 3 (ATF3)-a broad stress sensor-suppressed hormone-induced prostate carcinogenesis in mice. Although implantation of testosterone and estradiol (T+E2) pellets for 2 months in wild-type mice rarely induced prostatic intraepithelial neoplasia (PIN) in dorsal prostates (one out of eight mice), the loss of ATF3 led to the appearance of not only PIN but also invasive lesions in almost all examined animals. The enhanced carcinogenic effects of hormones on ATF3-deficient prostates did not appear to be caused by a change in estrogen signaling, but were more likely a consequence of elevated androgen signaling that stimulated differentiation of prostatic basal cells into transformation-preferable luminal cells. Indeed, we found that hormone-induced lesions in ATF3-knockout mice often contained cells with both basal and luminal characteristics, such as p63(+) cells (a basal-cell marker) showing luminal-like morphology, or cells double-stained with basal (CK5(+)) and luminal (CK8(+)) markers. Consistent with these findings, low ATF3 expression was found to be a poor prognostic marker for prostate cancer in a cohort of 245 patients. Our results thus support that ATF3 is a tumor suppressor in prostate cancer.

A critical function of the telomere is to disguise chromosome ends from cellular recognition as double strand breaks, thereby preventing aberrant chromosome fusion events. Such chromosome end-to-end fusions are known to initiate genomic instability via breakage-fusion-bridge cycles. Telomere dysfunction and other forms of genomic assault likely result in misregulation of genes involved in growth control, cell death, and senescence pathways, lowering the threshold to malignancy and likely drive disease progression. Shortened telomeres and anaphase bridges have been reported in a wide variety of early precursor and malignant cancer lesions including those of the prostate. These findings are being extended using methods for the analysis of telomere fusions (decisive genetic markers for telomere dysfunction) specifically within human tissue DNA. Here we report that benign prostatic hyperplasia (BPH), high-grade prostatic intraepithelial neoplasia (PIN), and prostate cancer (PCa) prostate lesions all contain similarly high frequencies of telomere fusions and anaphase bridges. Tumor-adjacent, histologically normal prostate tissue generally did not contain telomere fusions or anaphase bridges as compared to matched PCa tissues. However, we found relatively high levels of telomerase activity in this histologically normal tumor-adjacent tissue that was reduced but closely correlated with telomerase levels in corresponding PCa samples. Thus, we present evidence of high levels of telomere dysfunction in BPH, an established early precursor (PIN) and prostate cancer lesions but not generally in tumor adjacent normal tissue. Our results suggest that telomere dysfunction may be a common gateway event leading to genomic instability in prostate tumorigenesis. ., (© 2015 Wiley Periodicals, Inc.)

Purpose: The most important clinical significance of an isolated high-grade prostatic intraepithelial neoplasia (HGPIN) diagnosis is the risk of missed prostate cancer (PCa) in subsequent biopsies. Because most patients with HGPIN do not harbor or develop PCa, clinical, pathological, or molecular markers that predict of PCa risk are of clinical significance., Materials and Methods: Overall, 155 men with a diagnosis of isolated HGPIN, which was based on the results of extended biopsy, and who underwent at least one repeat biopsy were analyzed for ERG oncoprotein (ERG) expression and clinicopathological parameters to determine the risk of finding PCa in subsequent biopsies., Results: Of 155 patients diagnosed with HGPIN on initial biopsy, 39 (25%) had PCa on subsequent biopsies. For men with only one repeat biopsy, the cancer detection rate was 22%. Most (54%) PCas were detected in≤6 months of rebiopsy. ERG expression was present in 15 patients with HGPIN (9.6%). Patients with ERG expression in HGPIN were more likely to have PCa in repeat biopsy, with 9 (60%) ERG-positive and 30 (21%) of ERG-negative patients having PCa (P = 0.001). Multifocal involvement (P = 0.0001), cribriform morphology (P = 0.004), and bilaterality (P = 0.0075) of HGPIN were other significant risk factors. On multivariable analysis, only the presence of ERG positivity and multifocality remained significant parameters in detecting PCa on a repeat biopsy. The presence of ERG-negative focal HGPIN involving one core, which accounted for 46% of patients, had minimal (16%) PCa risk on subsequent biopsy. In total, 8 patients (89%) ERG-positive HGPIN had PCa identified at identical sites on subsequent biopsy, of which 5 (71%) were ERG positive., Conclusions: The status of ERG expression in HGPIN along with other histological parameters stratifies patients into low- and high-risk groups for having PCa on subsequent biopsy. Our results further support molecular characterization of HGPIN as a means to improve risk stratification and optimize surveillance strategies., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Emerging evidence has demonstrated the critical roles for both androgen and Wnt pathways in prostate tumorigenesis. A recent integrative genomic analysis of human prostate cancers (PCas) has revealed a unique enrichment of androgen and Wnt signaling in early-onset PCas, implying their clinical significance in the disease. Additionally, interaction between the androgen receptor (AR) and β-catenin has long been detected in PCa cells. However, the consequence of this interaction in prostate tumorigenesis is still unknown. Because mutations in adenomatous polyposis coli, β-catenin and other components of the destruction complex are generally rare in PCas, other mechanisms of aberrant Wnt signaling activation have been speculated. To address these critical questions, we developed Ctnnb1(L(ex3)/+)/R26hAR(L/+):PB-Cre4 mice, in which transgenic AR and stabilized β-catenin are co-expressed in prostatic epithelial cells. We observed accelerated tumor development, aggressive tumor invasion and a decreased survival rate in Ctnnb1(L(ex3)/+)/R26hAR(L/+):PB-Cre4 compound mice compared with age-matched Ctnnb1(L(ex3)/+):PB-Cre4 littermate controls, which only have stabilized β-catenin expression in the prostate. Castration of the above transgenic mice resulted in significant tumor regression, implying an essential role of androgen signaling in tumor growth and maintenance. Implantation of the prostatic epithelial cells isolated from the transgenic mice regenerated prostate intraepithelial neoplasias and prostatic adenocarcinoma lesions. Microarray analyses of transcriptional profiles showed more robust enrichment of known tumor- and metastasis-promoting genes: Spp1, Egr1, c-Myc, Sp5, and Sp6 genes, in samples isolated from Ctnnb1(L(ex3)/+)/R26hAR(L/+):PB-Cre4 compound mice than those from Ctnnb1(L(ex3)/+):PB-Cre4 and R26hAR(L/+):PB-Cre4 littermate controls. Together, these data demonstrate a confounding role of androgen signaling in β-catenin-initiated oncogenic transformation in prostate tumorigenesis.

Ethinylestradiol (EE) is an endocrine disruptor (ED) which acts as an oestrogen agonist; this compound is known as an oral contraceptive. Male and female rodents exposed to EE during critical time points of development, such as in the prenatal period, show alterations in their reproductive tract during adulthood. Few studies have placed an emphasis on the effects of EE during ageing. Thus, this study had as it's objective the analysis of the morphological and immunohistochemical effects of exposure to EE in the prenatal period on ventral male prostate and female prostate of gerbils (Meriones unguiculatus) during ageing. The animals were exposed to EE (15 μg/kg/day) during the 18-22th days of prenatal life (EE/PRE group), and the analyses were performed when the male and female reached 12 months of age. Our results showed an increase in the development of prostatic intraepithelial neoplasia (PIN), which was observed in the male and female prostate of EE/PRE groups. Immunohistochemistry showed a rise in prostatic epithelial and basal cells immunoreactivity, respectively, and to AR and p63 in the male EE/PRE. There were alterations in the morphological pattern of the prostatic glands and increase in predisposition to emergence of prostatic lesions of both sexes during ageing. Despite male and female having been exposed to the same doses of EE, the "exposure to EE promoted modifications" more accentuated in the male prostate. Thus the male gland is more sensitive to the action of this synthetic oestrogen than the female prostate., (© 2016 The Authors. International Journal of Experimental Pathology © 2016 International Journal of Experimental Pathology.)

LINE-1 (L1) retrotransposons are a source of endogenous reverse transcriptase (RT) activity, which is expressed as part of the L1-encoded ORF2 protein (L1-ORF2p). L1 elements are highly expressed in many cancer types, while being silenced in most differentiated somatic tissues. We previously found that RT inhibition reduces cell proliferation and promotes differentiation in neoplastic cells, indicating that high endogenous RT activity promotes cancer growth. Here we investigate the expression of L1-ORF2p in several human types of cancer.We have developed a highly specific monoclonal antibody (mAb chA1-L1) to study ORF2p expression and localization in human cancer cells and tissues.We uncover new evidence for high levels of L1-ORF2p in transformed cell lines and staged epithelial cancer tissues (colon, prostate, lung and breast) while no or only basal ORF2p expression was detected in non-transformed cells. An in-depth analysis of colon and prostate tissues shows ORF2p expression in preneoplastic stages, namely transitional mucosa and prostate intraepithelial neoplasia (PIN), respectively.Our results show that L1-ORF2p is overexpressed in tumor and in preneoplastic colon and prostate tissues; this latter finding suggests that ORF2p could be considered as a potential early diagnostic biomarker.

Prostate Cancer (PCa) holds the second place in terms of cancer-related mortality rate among men. The Notch signalling pathway regulates the proliferation and differentiation in embryonic and adult tissues and determines the cell fate. The body of knowledge in the present literature is currently controversial about the effect of the Notch pathway on prostatic cancer. Therefore, the present study aimed to examine the immunolocalization and expression levels of Notch1-4, Jagged1-2, Delta, HES1 and HES5 from among the members of the Notch signalling pathway in tissues of normal, prostatic intraepithelial neoplasia (PIN) and malignant prostate. The current study included a sample of 20 patients with localised prostatic adenocarcinoma, 18 patients with high grade PIN (H-PIN) and 18 normal prostatic tissue. Immunolocalisations of Notch1, 2, 3, 4, Jagged1, 2, Delta, HES1 and HES5 were identified through the immunohistochemical method. The findings of the present study showed that all in-scope members of the Notch signalling pathway were localised in PIN structures to a greater extent than in other tissues and from amongst these members, specifically Notch1, Notch4, Jagged1 and HES1 were at more significant levels. Consequently, the findings of the present study may indicate that the Notch signalling pathway can play a role especially in the formation of PIN structures.

Exposure of humans to bisphenol A (BPA) is widespread and continuous. The effects of protracted exposure to BPA on the adult prostate have not been studied. We subjected Noble rats to 32 weeks of BPA (low or high dose) or 17β-estradiol (E2) in conjunction with T replenishment. T treatment alone or untreated groups were used as controls. Circulating T levels were maintained within the physiological range in all treatment groups, whereas the levels of free BPA were elevated in the groups treated with T+low BPA (1.06 ± 0.05 ng/mL, P < .05) and T+high BPA (10.37 ± 0.43 ng/mL, P < .01) when compared with those in both controls (0.1 ± 0.05 ng/mL). Prostatic hyperplasia, low-grade prostatic intraepithelial neoplasia (PIN), and marked infiltration of CD4+ and CD8+ T cells into the PIN epithelium (P < .05) were observed in the lateral prostates (LPs) of T+low/high BPA-treated rats. In contrast, only hyperplasia and high-grade PIN, but no aberrant immune responses, were found in the T+E2-treated LPs. Genome-wide transcriptome analysis in LPs identified differential changes between T+BPA vs T+E2 treatment. Expression of multiple genes in the regulatory network controlled by hepatocyte nuclear factor 4α was perturbed by the T+BPA but not by the T+E2 exposure. Collectively these findings suggest that the adult rat prostate, under a physiologically relevant T environment, is susceptible to BPA-induced transcriptomic reprogramming, immune disruption, and aberrant growth dysregulation in a manner distinct from those caused by E2. They are more relevant to our recent report of higher urinary levels BPA found in patients with prostate cancer than those with benign disease.

Impaired Ca2+ signaling in prostate cancer contributes to several cancer hallmarks, such as enhanced proliferation and migration and a decreased ability to induce apoptosis. Na+ influx via transient receptor potential melastatin 4 channel (TRPM4) can reduce store-operated Ca2+ entry (SOCE) by decreasing the driving force for Ca2+. In patients with prostate cancer, gene expression of TRPM4 is elevated. Recently, TRPM4 was identified as a cancer driver gene in androgen-insensitive prostate cancer.We investigated TRPM4 protein expression in cancer tissue samples from 20 patients with prostate cancer. We found elevated TRPM4 protein levels in prostatic intraepithelial neoplasia (PIN) and prostate cancer tissue compared to healthy tissue. In primary human prostate epithelial cells (hPEC) from healthy tissue and in the androgen-insensitive prostate cancer cell lines DU145 and PC3, TRPM4 mediated large Na+ currents. We demonstrated significantly increased SOCE after siRNA targeting of TRPM4 in hPEC and DU145 cells. In addition, knockdown of TRPM4 reduced migration but not proliferation of DU145 and PC3 cells. Taken together, our data identify TRPM4 as a regulator of SOCE in hPEC and DU145 cells, demonstrate a role for TRPM4 in cancer cell migration and suggest that TRPM4 is a promising potential therapeutic target.

Background: There is limited information about the clinical and biological significance of prostate specific G protein coupled receptor (PSGR) in prostate cancer (PCa) initiation and progression. Here, we evaluated the expression of PSGR protein, studied its diagnostic and prognostic value in PCa, and also explored its role in cancer cell growth and invasion., Methods: The expression of PSGR in paired adjacent normal prostate, high grade prostatic intraepithelial neoplasia (PIN), and PCa were determined by immunohistochemistry on tissue microarrays constructed from 150 radical prostatectomy specimens. The effects of PSGR on PCa cell growth and invasion were investigated using human PCa cell lines., Results: Membranous and cytoplasmic PSGR staining was observed at luminal epithelial cells of prostate. PSGR protein expression was significantly higher in PIN compared to normal prostate. Interestingly, the expression of PSGR decreased as PIN progressed to PCa. Low PSGR expression in PCa was associated with high Gleason score, and poor overall survival. Activated PSGR increased cancer cell invasive ability, but retarded cell growth. PSGR did not affect mTOR activity, but suppressed P70 S6 kinase activity., Conclusions: PSGR may participate in PCa progression through affecting cell proliferation and invasion. High expression of PSGR in PIN may implicate its role in early neoplastic transformation of PCa. Low expression of PSGR in PCa may serve as a potential indicator for poor prognosis.

Interleukin-6 (IL-6) has been associated with the development of prostate cancer. The aim of the study was to clarify whether IL-6 expression in prostate tissue could be a useful marker in differentiation of prostate diseases in small foci by pathologist visual scoring. Archival paraffin-embedded specimens of benign prostate hyperplasia (BPH), high-grade prostatic intraepithelial neoplasia (PIN), prostatitis and prostate adenocarcinoma were studied by immunohistochemistry with a mouse monoclonal antibody IL-6 using the streptavidin-biotin method. Significantly, lower IL-6 immunoreactivity was observed in normal epithelial cells (p=0.000) and basal cells (p=0.000) in the samples of prostate adenocarcinoma in comparison to the samples with BPH, PIN and prostatitis. There was no significant difference in IL-6 expression in malignant and premalignant cells (p=0.814) as well as in stromal cells among the four diagnoses (p=0.22). IL-6 was expressed in normal epithelial cells, premalignant epithelial cells and malignant epithelial cells as well as in stromal cells. However, in our research IL-6 was of limited utility as a single marker for differential diagnosis of the prostate diseases in small foci needle biopsy by pathologist visual scoring. The standardization of immunohistochemical (IHC) staining protocol for IL-6 is required to determine IL-6 expression in order to avoid possible misinterpretation of the IHC results., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

The differential diagnosis for atypical cribriform lesions of the prostate has become increasingly complex and includes intraductal carcinoma of the prostate, high-grade prostatic intraepithelial neoplasia, and atypical intraductal proliferations. In this review, we summarize the morphologic and molecular features and significance of intraductal carcinoma of the prostate. We also summarize our institution's strategy for reporting and treatment recommendations for intraductal carcinoma of the prostate.

Prostate cancer is the most common cancer among men in the Unites States. The cytokine IL-6 activates several prostate cancer pathways, but its upstream trans-signaling pathway remains poorly understood. In this study, we evaluated the role of IL-6 in PDCD4 gene expression and how the microRNA miR-21 regulates this process in prostate cancer cell lines PC-3 and LNCaP. The expression pattern of PDCD4 from samples from human prostate cancer, precancerous lesions, and benign prostatic hyperplasia was investigated by immunohistochemistry. PDCD4 transcription and translation were detected by quantitative real-time PCR (qRT-PCR) and Western blot analysis, respectively. The targeted modulation of PDCD4 by miR-21 was analyzed in PC-3 and LNCaP cells, and the effect of IL-6 on the expression of PDCD4 was studied in vitro. PDCD4 expression in samples from the 3 tissue types progressively increased, and the expression levels of PDCD4 and prostate-specific antigen were negatively correlated. The levels of PDCD4 mRNA and protein in PC-3 and LNCaP cells transfected with anti-miR-21 constructs were lower than those in control cells. The expression of PDCD4 was inhibited by IL-6, but this effect was weakened in cell lines with low expression of miR-21. Our study demonstrates that the regulation of PDCD4 by miR-21 is targeted and IL-6 inhibits expression of the PDCD4 gene in PC-3 and LNCaP cells through the targeted function of miR-21 on PDCD4. These findings support the feasibility of future efforts for diagnosis and gene therapy for prostate cancer that are based on IL-6, miR-21, and PDCD4.

The phosphatase and tensin homolog (PTEN) and the zinc finger homeobox 3 (ZFHX3)/AT-motif binding factor 1 (ATBF1) genes have been established as tumor suppressor genes in prostate cancer by their frequent deletions and mutations in human prostate cancer and by the formation of mouse prostatic intraepithelial neoplasia (mPIN) or tumor by their deletions in mouse prostates. However, whether ZFHX3/ATBF1 deletion together with PTEN deletion facilitates prostatic tumorigenesis is unknown. In this study, we simultaneously deleted both genes in mouse prostatic epithelia and performed histological and molecular analyses. While deletion of one Pten allele alone caused low-grade (LG) mPIN as previously reported, concurrent deletion of Zfhx3/Atbf1 promoted the progression to high-grade (HG) mPIN or early carcinoma. Zfhx3/Atbf1 and Pten deletions together increased cell proliferation, disrupted the smooth muscle layer between epithelium and stroma, and increased the number of apoptotic cells. Deletion of both genes also accelerated the activation of Akt and Erk1/2 oncoproteins. These results suggest an additive effect of ZFHX3/ATBF1 and PTEN deletions on the development and progression of prostate neoplasia., (Copyright © 2015 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.)

ERG and PTEN biomarkers are increasingly being analyzed on prostate core biopsies (NBXs); ERG as a marker of clonality and number of separately arising tumor foci and PTEN for prognostic information. Yet, in patients with multiple biopsy cores positive for cancer (PCa), there is no standardized approach for interrogation of these biomarkers in terms of the number of positive cores to evaluate. A total of 194 NBX cases containing more than one positive core with cancer were evaluated for ERG overexpression and PTEN loss by immunostaining (immunohistochemistry) of all positive cores. ERG overexpression or PTEN loss in at least one cancer core was present in 111 (57%) and 69 (36%) cases respectively. ERG overexpression was significantly associated with PTEN loss (P < .0001), and PTEN loss was associated with a high Gleason score (P < .0001). Inter- and intra-tumor core staining heterogeneity for ERG overexpression occurred in 42% and 5% cases and for PTEN loss both intra- and inter-tumor core heterogeneity was 68%. PTEN staining was highly discordant between PCa sites regardless of laterality. When the Gleason score was non-uniform across PCa sites, the combination of cores showing the highest Gleason score and largest tumor volume provided the best representation of ERG overexpression (92%) and PTEN loss (98%). When grades were uniform across cancer sites, the highest tumor volume core was generally representative of ERG overexpression (90%) but was less representative for PTEN loss (76%). Our results suggest that knowledge of this heterogeneity is critical for developing optimal yet cost-effective strategies to identify these underlying molecular abnormalities., (Copyright © 2015 Elsevier Inc. All rights reserved.)