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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.)

Aberrant phosphoinositide 3-kinase (PI3K), mitogen-activated protein kinase (MAPK) and WNT signalling are emerging as key events in the multistep nature of prostate tumourigenesis and progression. Here, we report a compound prostate cancer murine model in which these signalling pathways cooperate to produce a more aggressive prostate cancer phenotype. Using Cre-LoxP technology and the probasin promoter, we combined the loss of Pten (Pten fl/fl ), to activate the PI3K signalling pathway, with either dominant stabilized β-catenin [Catnb +/lox(ex3) ] or activated K-RAS (K-Ras +/V12 ) to aberrantly activate WNT and MAPK signalling, respectively. Synchronous activation of all three pathways (triple mutants) significantly reduced survival (median 96 days) as compared with double mutants [median: 140 days for Catnb +/lox(ex3) Pten fl/fl ; 182 days for Catnb +/lox(ex3) K-Ras +/V12 ; 238 days for Pten fl/fl K-Ras +/V12 ], and single mutants [median: 383 days for Catnb +/lox(ex3) ; 407 days for Pten fl/fl ], reflecting the accelerated tumourigenesis. Tumours followed a stepwise progression from mouse prostate intraepithelial neoplasia to invasive adenocarcinoma, similar to that seen in human disease. There was significantly elevated cellular proliferation, tumour growth and percentage of invasive adenocarcinoma in triple mutants as compared with double mutants and single mutants. Triple mutants showed not only activated AKT, extracellular-signal regulated kinase 1/2, and nuclear β-catenin, but also significantly elevated signalling through mechanistic target of rapamycin complex 1 (mTORC1). In summary, we show that combined deregulation of the PI3K, MAPK and WNT signalling pathways drives rapid progression of prostate tumourigenesis, and that deregulation of all three pathways results in tumours showing aberrant mTORC1 signalling. As mTORC1 signalling is emerging as a key driver of androgen deprivation therapy resistance, our findings are important for understanding the biology of therapy-resistant prostate cancer and identifying potential approaches to overcome this. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd., (Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Background: Although men on active surveillance for prostate cancer (PCa) may benefit from intervention with 5α-reductase inhibitors (5-ARIs), it has not been resolved whether 5-ARIs are effective for delaying disease progression and, if so, whether specific patients are more likely to benefit., Objective: To identify molecular features predictive of patient response to 5-ARIs., Design, Setting, and Participants: Nkx3.1 mutant mice, a model of early-stage PCa, were treated with the 5-ARI finasteride, and histopathological and molecular analyses were performed. Cross-species computational analyses were used to compare expression profiles for treated mice with those of patients who had received 5-ARIs before prostatectomy., Intervention: Finasteride administered to Nkx3.1 mutant mice. 5-ARI-treated patient specimens obtained retrospectively., Outcome Measurements and Statistical Analysis: Endpoints in mice included histopathology, immunohistochemistry, and molecular profiling. GraphPad Prism software, R-studio, and Matlab were used for statistical and data analyses., Results and Limitations: Finasteride treatment of Nkx3.1 mutant mice resulted in a significant reduction in prostatic intraepithelial neoplasia (PIN), as evident from histopathological and expression profiling analyses. Cross-species computational analysis comparing finasteride-treated mice with two independent 5-ARI-treated patient cohorts showed that reduced NKX3.1 expression is predictive of response to 5-ARI. A limitation of the study is that these retrospective human cohorts have relatively few patients with limited clinical outcome data. Future prospective clinical trials are needed to validate whether stratifying patients on the basis of NKX3.1 expression improves the benefit of 5-ARIs during active surveillance., Conclusions: This co-clinical study implicates NKX3.1 status as a predictor of response to 5-ARIs, and suggests that molecular features, including NKX3.1 expression, may help to identify PCa patients most likely to benefit from 5-ARIs during active surveillance., Patient Summary: The aim of precision cancer prevention is to tailor interventions on the basis of individualized patient characteristics. We propose that patients with low NKX3.1 expression are optimal candidates for intervention with 5α-reductase inhibitors as an adjunct to active surveillance., (Copyright © 2017 European Association of Urology. Published by Elsevier B.V. All rights reserved.)

Increased polyamine synthesis is known to play an important role in prostate cancer. We aimed to explore its functional significance in prostate tumor initiation and its link to androgen receptor (AR) signaling. For this purpose, we generated a new cell line derived from normal epithelial prostate cells (RWPE-1) with overexpression of ornithine decarboxylase (ODC) and used it for in vitro and in vivo experiments. We then comprehensively analyzed the expression of the main metabolic enzymes of the polyamine pathway and spermine abundance in 120 well-characterized cases of human prostate cancer and high-grade prostate intraepithelial neoplasia (HGPIN). Herein, we show that the ODC-overexpressing prostate cells underwent malignant transformation, revealing that ODC is sufficient for de novo tumor initiation in 94% of injected mice. This oncogenic capacity was acquired through alteration of critical signaling networks, including AR, EIF2, and mTOR/MAPK. RNA silencing experiments revealed the link between AR signaling and polyamine metabolism. Human prostate cancers consistently demonstrated up-regulation of the main polyamine enzymes analyzed (ODC, polyamine oxidase, and spermine synthase) and reduction of spermine. This phenotype was also dominant in HGPIN, rendering it a new biomarker of malignant transformation. In summary, we report that ODC plays a key role in prostate tumorigenesis and that the polyamine pathway is altered as early as HGPIN., (Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

High-grade prostatic intraepithelial neoplasia (HGPIN) is widely believed to represent a precursor to invasive prostatic adenocarcinoma. However, recent molecular studies have suggested that retrograde spread of invasive adenocarcinoma into pre-existing prostatic ducts can morphologically mimic HGPIN. Thus, previous molecular studies characterizing morphologically identified HGPIN occurring in radical prostatectomies or needle biopsies with concurrent invasive carcinoma may be partially confounded by intraductal spread of invasive tumor. To assess ERG and PTEN status in HGPIN foci likely to represent true precursor lesions in the prostate, we studied isolated HGPIN occurring without associated invasive adenocarcinoma in cystoprostatectomies performed at Johns Hopkins between 2009 and 2014. Of 344 cystoprostatectomies, 33% (115/344) contained invasive prostatic adenocarcinoma in the partially submitted prostate (10 blocks/case on average) and were excluded from the study. Of the remaining cases without sampled cancer, 32% (73/229) showed 133 separate foci of HGPIN and were immunostained for ERG and PTEN using genetically validated protocols. Of foci of HGPIN with evaluable staining, 7% (8/107) were positive for ERG. PTEN loss was not seen in any HGPIN lesion (0/88). Because these isolated HGPIN foci at cystoprostatectomy are unlikely to represent retrograde spread of invasive tumor, our study suggests that ERG rearrangement, but not PTEN loss, is present in a minority of potential neoplastic precursor lesions in the prostate., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Fermented brown rice and rice bran with Aspergillus oryzae (FBRA) is considered to have the potential to prevent chemically-induced carcinogenesis in multiple organs of rodents. In the present study, we evaluated the possible chemopreventive effects of FBRA against prostate tumorigenesis. Six-week-old male rats of the transgenic rat for adenocarcinoma of prostate (TRAP) strain were fed diets containing 5% or 10% FBRA for 15 weeks. Animals were sacrificed at 21 weeks of age, and the ventral and lateral prostate were removed for histopathological evaluation and immunoblot analyses. FBRA decreased the incidence of adenocarcinoma in the lateral prostate and suppressed the progression of prostate carcinogenesis. Treatment with FBRA induced apoptosis and inhibited cell proliferation in histologically high-grade prostatic intraepithelial neoplasias. Phospho-AMP-activated kinase α (Thr172) was up-regulated in the prostate of rats fed the diet supplemented with FBRA. These results indicate that FBRA controls tumor growth by activating pathways responsive to energy deprivation and suggest that FBRA has translational potential for the prevention of human prostate cancer.

Membrane-associated guanylate kinase, WW and PDZ domain-containing protein 2 (MAGI-2) is a scaffolding protein that links cell adhesion molecules, receptors, and signaling molecules to the cytoskeleton and maintains the architecture of cell junctions. MAGI-2 gene rearrangements have recently been described in prostate cancer. We studied the immunohistochemical expression of MAGI-2 protein in prostate tissue. Seventy-eight radical prostatectomies were used to construct 3 tissue microarrays consisting of 512 cores, including benign tissue, benign prostatic hyperplasia, high-grade prostatic intraepithelial neoplasia (HGPIN), and adenocarcinoma, Gleason patterns 3 to 5. Immunohistochemistry for phosphatase and tensin homologue (PTEN) and double-stain MAGI-2/p63 was performed and analyzed by visual and image analysis, the latter as percent of analyzed area (%AREA), and mean optical density multiplied by %AREA (STAIN). By visual and image analysis, MAGI-2 was significantly higher in adenocarcinoma and HGPIN compared with benign (benign versus HGPIN P < .001; benign versus adenocarcinoma, P < .001). HGPIN and adenocarcinoma did not significantly differ by either modality. Using visual intensity to distinguish benign tissue and adenocarcinoma, a receiver operating curve yielded an area under the curve of 0.902. A STAIN threshold of 1470 yielded a sensitivity of 0.66 and specificity of 0.96. There was a significant correlation between PTEN and MAGI-2 staining for normal and benign prostatic hyperplasia, but this was lost in HGPIN and cancer. We conclude that MAGI-2 immunoreactivity is elevated in prostate cancer and HGPIN compared with normal tissue, and suggest that MAGI-2 may contribute to prostate carcinogenesis. This is the first report of MAGI-2 staining by immunohistochemistry in prostate cancer., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Tocopherols, the major forms of vitamin E, exist as alpha-tocopherol (α-T), β-T, γ-T and δ-T. The cancer preventive activity of vitamin E is suggested by epidemiological studies, but recent large-scale cancer prevention trials with high dose of α-T yielded disappointing results. Our hypothesis that other forms of tocopherols have higher cancer preventive activities than α-T was tested, herein, in a novel prostate carcinogenesis model induced by 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP), a dietary carcinogen, in the CYP1A-humanized (hCYP1A) mice. Treatment of hCYP1A mice with PhIP (200 mg/kg b.w., i.g.) induced high percentages of mouse prostatic intraepithelial neoplasia (mPIN), mainly in the dorsolateral glands. Supplementation with a γ-T-rich mixture of tocopherols (γ-TmT, 0.3% in diet) significantly inhibited the development of mPIN lesions and reduced PhIP-induced elevation of 8-oxo-deoxyguanosine, COX-2, nitrotyrosine, Ki-67 and p-AKT, and the loss of PTEN and Nrf2. Further studies with purified δ-T, γ-T or α-T (0.2% in diet) showed that δ-T was more effective than γ-T or α-T in preventing mPIN formations and p-AKT elevation. These results indicate that γ-TmT and δ-T could be effective preventive agents of prostate cancer., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

The tumor microenvironment plays a critical role in cancer progression, but the precise mechanisms by which stromal cells influence the epithelium are poorly understood. Here we show that p62 levels were reduced in the stroma of several tumors and that its loss in the tumor microenvironment or stromal fibroblasts resulted in increased tumorigenesis of epithelial prostate cancer cells. The mechanism involves the regulation of cellular redox through an mTORC1/c-Myc pathway of stromal glucose and amino acid metabolism, resulting in increased stromal IL-6 production, which is required for tumor promotion in the epithelial compartment. Thus, p62 is an anti-inflammatory tumor suppressor that acts through the modulation of metabolism in the tumor stroma., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Early diagnosis of prostate cancer (CaP) can be addressed by studying prostatic intraepithelial neoplasia (PIN) as precancer (high-grade PIN or HGPIN). This article attempts to analyze the diagnostic role of telomerase as an early marker of carcinogenesis., Methods: Complex urological patient evaluation and assessment of telomerase activity., Results: Out of 92 patients 44% were diagnosed with CaP, 49% with low-grade PIN (LGPIN) in association with benign prostatic hyperplasia (BPH), and 7% with HGPIN in association with BPH. Active telomerase (AT) in prostate biopsy specimens was detected in 98% of patients with CaP, in 33% of patients with HGPIN, and in 20% of patients with LGPIN. In the event of simultaneous detection of AT and PIN in initial prostate biopsy specimens, further monitoring for 0.5-4.0 years revealed CaP development in 50-56% of cases. Further follow-up of patients with PIN and absent telomerase activity in initial biopsy specimens did not demonstrate the development of CaP. The PSA level was significantly higher in patients with active telomerase in the prostate tissue than in telomerase negative patients., Conclusions: Telomerase activity in the prostate tissue increases the risk of CaP development in patients with PIN. Detection of telomerase activity in prostate biopsy specimens from patients with PIN enables selection of a group of patients with high risk of CaP development and reduction of the number of prostate biopsies performed in other patients., (© 2014 Wiley Periodicals, Inc.)

Background: Accumulating evidence demonstrates high levels of aldehyde dehydrogense (ALDH) activity in human cancer types, in part, because of its association with cancer stem cells. Whereas ALDH1A1 and ALDH7A1 isoforms were reported to associate with prostate tumorigenesis, whether other ALDH isoforms are associated with prostate cancer (PC) remains unclear., Methods: ALDH3A1 expression was analysed in various PC cell lines. Xenograft tumours and 54 primary and metastatic PC tumours were stained using immunohistochemistry for ALDH3A1 expression., Results: In comparison with the non-stem counterparts, a robust upregulation of ALDH3A1 was observed in DU145-derived PC stem cells (PCSCs). As DU145 PCSCs produced xenograft tumours with more advanced features compared with those derived from DU145 cells, higher levels of ALDH3A1 were detected in the former; a dramatic elevation of ALDH3A1 occurred in DU145 cell-derived lung metastasis compared with local xenograft tumours. Furthermore, while ALDH3A1 was not observed in prostate glands, ALDH3A1 was clearly present in PIN, and further increased in carcinomas. In comparison with the paired local carcinomas, ALDH3A1 was upregulated in lymph node metastatic tumours; the presence of ALDH3A1 in bone metastatic PC was also demonstrated., Conclusions: We report here the association of ALDH3A1 with PC progression.

Overexpression of the histone acetyltransferase p300 is implicated in the proliferation and progression of prostate cancer, but evidence of a causal role is lacking. In this study, we provide genetic evidence that this generic transcriptional coactivator functions as a positive modifier of prostate tumorigenesis. In a mouse model of PTEN deletion-induced prostate cancer, genetic ablation of p300 attenuated expression of the androgen receptor (AR). This finding was confirmed in human prostate cancer cells in which PTEN expression was abolished by RNA interference-mediated attenuation. These results were consistent with clinical evidence that the expression of p300 and AR correlates positively in human prostate cancer specimens. Mechanistically, PTEN inactivation increased AR phosphorylation at serine 81 (Ser81) to promote p300 binding and acetylation of AR, thereby precluding its polyubiquitination and degradation. In support of these findings, in PTEN-deficient prostate cancer in the mouse, we found that p300 was crucial for AR target gene expression. Taken together, our work identifies p300 as a molecular determinant of AR degradation and highlights p300 as a candidate target to manage prostate cancer, especially in cases marked by PTEN loss., (©2014 AACR.)

Objective: The objective of this study was to investigate the significance of the activated Akt-mammalian target of rapamycin (Akt-mTOR) signaling pathway in the progression of prostate cancer., Materials and Methods: The expression levels of Akt, phosphorylated Akt (p-Akt), mTOR and phosphorylated mTOR (p-mTOR) in 175 prostate specimens, including 61 normal prostate tissues as a control, 24 high-grade prostatic intraepithelial neoplasias (HGPINs) and 90 clinically localized prostate cancers, were evaluated by immunohistochemical staining. p-Akt and p-mTOR ratios, which were defined as the expression level of p-Akt in relation to that of Akt and the expression level of p-mTOR in relation to that of mTOR, respectively, in these specimens were calculated., Results: Expression levels of all four molecules, including Akt, p-Akt, mTOR and p-mTOR, were significantly greater in the HGPIN group compared with the normal control and prostate cancer groups. Furthermore, the p-Akt ratio in the prostate cancer group was significantly lower than that in the HGPIN group, while there was no significant difference in the p-mTOR ratio between the HGPIN and prostate cancer groups. In the prostate cancer group, no significant relationships were observed between major clinicopathological parameters and the expression levels as well as the ratios of p-Akt or p-mTOR., Conclusions: The Akt-mTOR signaling pathway may play a limited role in the progression of prostate cancer., (2014 S. Karger AG, Basel.)

Background: Alpha methylacyl-CoA racemase (AMACR) is a useful diagnostic marker for prostate adenocarcinoma. However, its usefulness has not been fully validated in Japanese patients. The aim of this study was to evaluate the diagnostic utility of AMACR in prostate needle biopsy examination in Japanese patients., Methods: A total of 119 prospective consecutive prostate needle biopsy specimens (680 cores) obtained from Japanese patients were examined. Sixty patients had adenocarcinoma (adenocarcinoma, 160 cores; benign, 204 cores), 14 patients had high-grade prostatic intraepithelial neoplasia (HGPIN; 19 cores), and 45 patients did not have any neoplastic lesions (297 cores). AMACR expression was scored semi-quantitatively as 0 (no expression), 1+ (partial and/or weak expression), or 2+ (strong, circumferential expression). The number of positively stained glands was counted., Results: 2+ AMACR expression was observed in 70.1% of adenocarcinoma cases and in 52.6% of HGPIN cases. Of the adenocarcinoma cases showing 2+ AMACR expression, 34.8% demonstrated a heterogeneous expression pattern, with 1-75% of AMACR-positive glands. Three hundred eighty-five of the benign glands with an adenocarcinoma component showed 2+ AMACR expression (35 cases, 94 cores). 2+ AMACR expression was observed in 67 non-neoplastic benign glands (9 cases, 19 cores)., Conclusions: The sensitivity and specificity of AMACR for the diagnosis of prostate adenocarcinoma and benign glands in Japanese patients are lower than those previously reported in Western countries. Pathologists should be cautious while interpreting AMACR expression pattern in Japanese patients., (Copyright © 2012 Wiley Periodicals, Inc.)

Tp53 mutations are common in human prostate cancer (CaP), occurring with a frequency of ∼30% and ∼70% in localized and metastatic disease, respectively. In vitro studies have determined several common mutations of Tp53 that have specific gain-of-function properties in addition to loss of function, including the ability to promote castration-resistant (CR) growth of CaP cells in some contexts. To date, a lack of suitable mouse models has prohibited investigation of the role played by Tp53 mutations in mediating CaP progression in vivo. Here, we describe the effects of conditional expression of a mutant Tp53 (Tp53(R270H); equivalent to the human hotspot mutant R273H) in the prostate epithelium of mice. Heterozygous "Tp53(LSL-R270H/+)" [129S4(Trp53(tm3Tyj))] and "Nkx3.1-Cre" [129S(Nkx3-1(tm3(cre)Mms))] mice with prostate-specific expression of the Tp53(R270H) mutation (p53(R270H/+) Nkx3.1-Cre mice) were bred onto an FVB/N background via speed congenesis to produce strain FVB.129S4(Trp53(tm3Tyj/wt)); FVB.129S(Nkx3-1(tm3(cre)Mms/wt)) and littermate genotype negative control mice. These mutant mice had significantly increased incidences of prostatic intraepithelial neoplasia (PIN) lesions, and these appeared earlier, compared with the Nkx3.1 haploinsufficient (Nkx3.1-Cre het) littermate mice, which did not express the Tp53 mutation. PIN lesions in these mice showed consistent progression and some developed into invasive adenocarcinoma with a high grade, sarcomatoid or epithelial-mesenchymal transition (EMT) phenotype. PIN lesions were similar to those seen in PTEN conditional knockout mice, with evidence of AKT activation concomitant with neoplastic proliferation. However, the invasive tumor phenotype is rarely seen in previously described mouse models of prostatic neoplasia. These data indicate that the Tp53(R270H) mutation plays a role in CaP initiation. This finding has not previously been reported. Further characterization of this model, particularly in a setting of androgen deprivation, should allow further insight into the mechanisms by which the Tp53(R270H) mutation mediates CaP progression.

Prostate cancer (PCa) is a major lethal malignancy in men, but the molecular events and their interplay underlying prostate carcinogenesis remain poorly understood. Epigenetic events and the upregulation of polycomb group silencing proteins including Bmi1 have been described to occur during PCa progression. Here, we found that conditional overexpression of Bmi1 in mice induced prostatic intraepithelial neoplasia, and elicited invasive adenocarcinoma when combined with PTEN haploinsufficiency. In addition, Bmi1 and the PI3K/Akt pathway were coactivated in a substantial fraction of human high-grade tumors. We found that Akt mediated Bmi1 phosphorylation, enhancing its oncogenic potential in an Ink4a/Arf-independent manner. This process also modulated the DNA damage response and affected genomic stability. Together, our findings demonstrate the etiological role of Bmi1 in PCa, unravel an oncogenic collaboration between Bmi1 and the PI3K/Akt pathway, and provide mechanistic insights into the modulation of Bmi1 function by phosphorylation during prostate carcinogenesis.

Caspases are essential initiators and executioners of apoptosis. Changes in their expression may contribute to the development of proliferative disorders such as cancer, by altering the death-proliferation homeostatic balance. The aim of this work was to analyze the expression of a broad panel of caspases at the epithelial level in human prostate tissues to assess possible prostatic disease-related alterations. We comparatively analyzed by immunohistochemistry the expression of pro-caspase-3, pro-caspase-8, pro-caspase-9, cleaved caspase-3, cleaved caspase-8, and caspase-7, in normal and pathologic (benign hyperplasic, premalignant [high-grade intraepithelial neoplasia], and cancerous [prostate cancer]) human prostate epithelium. Expression of caspases was correlated with clinicopathologic features, including preoperative prostate-specific antigen levels, Gleason scores, and biochemical progression. Percentage of positive samples for all the analyzed caspases decreased in prostate cancer versus normal prostate epithelium. The values obtained for benign prostatic hyperplasia and high-grade intraepithelial neoplasia more qualitatively resembled those of the prostate cancer group. Our results indicate that caspase expression in prostate malignant cells is reduced in a substantial number of patients and that such an alteration occurs in the premalignant stage. Loss of caspase expression could constitute a useful marker for prostate cancer diagnosis. Therapeutic approaches aimed to recover or enhance caspase expression might be effective against prostate cancer., (Copyright © 2012 Elsevier Inc. All rights reserved.)

MYC and phosphoinositide 3-kinase (PI3K)-pathway deregulation are common in human prostate cancer. Through examination of 194 human prostate tumors, we observed statistically significant co-occurrence of MYC amplification and PI3K-pathway alteration, raising the possibility that these two lesions cooperate in prostate cancer progression. To investigate this, we generated bigenic mice in which both activated human AKT1 and human MYC are expressed in the prostate (MPAKT/Hi-MYC model). In contrast to mice expressing AKT1 alone (MPAKT model) or MYC alone (Hi-MYC model), the bigenic phenotype demonstrates accelerated progression of mouse prostate intraepithelial neoplasia (mPIN) to microinvasive disease with disruption of basement membrane, significant stromal remodeling and infiltration of macrophages, B- and T-lymphocytes, similar to inflammation observed in human prostate tumors. In contrast to the reversibility of mPIN lesions in young MPAKT mice after treatment with mTOR inhibitors, Hi-MYC and bigenic MPAKT/Hi-MYC mice were resistant. Additionally, older MPAKT mice showed reduced sensitivity to mTOR inhibition, suggesting that additional genetic events may dampen mTOR dependence. Since increased MYC expression is an early feature of many human prostate cancers, these data have implications for treatment of human prostate cancers with PI3K-pathway alterations using mTOR inhibitors.

It is well established that protein kinase C (PKC) isozymes play distinctive roles in mitogenic and survival signaling as well as in cancer progression. PKCε, the product of the PRKCE gene, is up-regulated in various types of cancers including prostate, lung and breast cancer. To address a potential role for PKCs in prostate cancer progression we generated three mouse transgenic lines expressing PKCα, PKCδ, or PKCε in the prostate epithelium under the control of the rat probasin (PB) promoter. Whereas PB-PKCε and PB-PKCδ mice did not show any evident phenotype, PB-PKCε mice developed prostate hyperplasia as well as prostate intraepithelial neoplasia (PIN) that displayed enhanced phospho-Akt, phospho-S6, and phospho-Stat3 levels, as well as enhanced resistance to apoptotic stimuli. PKCε overexpression was insufficient to drive neoplastic changes in the mouse prostate. Notably, overexpression of PKCε by adenoviral means in normal immortalized RWPE-1 prostate cells confers a growth advantage and hyperactivation of Erk and Akt. Our results argue for a causal link between PKCε overexpression and prostate cancer development.

Background: There is growing evidence that inflammation is a causal factor in cancer, where pro-inflammatory cytokines such as IL-6, IL-1 or TNF-α could induce cellular proliferation by activation of NF-κB. This study focuses on the IL-6/ERK transduction pathway, its relationship with NF-κB, and the consequences of dysregulation in the development of prostate pathologies such as benign prostate hyperplasia (BPH), prostate intraepithelial neoplasia (PIN) and prostate cancer (PC)., Methods: Immunohistochemical and Western blot analyses for IL-6, gp-130, Raf-1, MEK-1, ERK-1, p-MEK, ERK-2, p-ERK, NF-κB/p-50 and NF-κB/p-65 were carried out in 20 samples of normal prostate glands, 35 samples of BPH, 27 samples with a diagnosis of PIN (low-grade PIN or high-grade PIN), and 95 samples of PC (23 with low, 51 with medium and 21 with high Gleason scores)., Results: Immunoreaction to IL-6, gp-130, ERK-1, ERK-2, p-ERK and NF-κB/p50 was found in the cytoplasm of epithelial cells in normal prostate samples; p-MEK was found in the nucleus of epithelial cells; but not expression to Raf-1, MEK-1 and NF-κB/p65. In BPH, all of these proteins were immunoexpressed, while there was increased immunoexpression of IL-6, gp-130, p-MEK, ERK-1, ERK-2 and NF-κB/p50 (cytoplasm). In PC, immunoexpression of IL-6 and gp-130 were similar to that found in BPH; while immunoexpression of Raf-1, MEK-1, p-MEK, ERK-1, ERK-2, p-ERK, NF-κB/p50 (nucleus and cytoplasm), and NF-κB/p65 (nucleus and cytoplasm) was higher than in BPH., Conclusion: Translocation of NF-κB to the nucleus in PC and high-grade PIN could be stimulated by the IL-6/ERK transduction pathway, but might also be stimulated by other transduction pathways, such as TNF-α/NIK, TNF/p38, IL-1/NIK or IL-1/p38. Activation of NF-κB in PC could regulate IL-6 expression. These transduction pathways are also related to activation of other transcription factors such as Elk-1, ATF-2 or c-myc (also involved in cell proliferation and survival). PC is a heterogeneous disease, where multiple transduction pathways might alter the apoptosis/proliferation balance. Significant attention should be give to the combination of novel agents directed towards inactivation of pro-inflammatory cytokines than can disrupt tumour cell growth.

Background: By alternative splicing the IGF-1 gene produces several different transcripts, including IGF-1Ec (MGF). The latter has been mainly associated with muscle regeneration processes., Methods: We used immunohistochemistry, RT-PCR, and Western analysis to show the expression status of MGF in prostate tissue and human prostate cell lines (HPrEC, PC-3, and LNCaP) and we studied the exogenous administration of the MGF peptide E on cellular proliferation using trypan blue and MTT assays, before and after the silencing of the IGF-1 receptor and insulin receptor (siRNA methods). The MGF-induced intracellular activation was examined by Western analysis of the active forms of ERK1/2 and Akt., Results: We documented that MGF is overexpressed in human prostate cancer (PCa) tissues and in human PC-3 and LNCaP cells. Notably, MGF expression was remarkably higher in PCa and prostatic intraepithelial neoplasia (PIN) than normal prostate tissues, while the normal prostate epithelial cells (HPrEC) did not express MGF. Exogenous administration of a synthetic MGF E peptide stimulated the PCa cell growth and activated ERK1/2 phosphorylation without affecting Akt phosphorylation. IGF-1R or insulin receptor (IR) silencing did not affect the mitogenic activity and intracellular signaling of the MGF E peptide in these PCa cells., Conclusions: These data suggest the possible implication of MGF E peptide in cancer biology, implying a preferential MGF expression in PCa tissues and cells. This preferential IGF-1 mRNA expression generates the MGF E peptide that possesses mitogenic activity through mechanisms independent of IGF-1R, IR, and hybrid IGF-1R/IR., (2010. (c) 2010 Wiley-Liss, Inc.)

Recent work has shown that ablation of p110beta, but not p110alpha, markedly impairs tumorigenesis driven by loss of phosphatase and tensin homolog (PTEN) in the mouse prostate. Other laboratories have reported complementary data in human prostate tumor lines, suggesting that p110beta activation is necessary for tumorigenesis driven by PTEN loss. Given the multiple functions of PTEN, we wondered if p110beta activation also is sufficient for tumorigenesis. Here, we report that transgenic expression of a constitutively activated p110beta allele in the prostate drives prostate intraepithelial neoplasia formation. The resulting lesions are similar to, but are clearly distinct from, the ones arising from PTEN loss or Akt activation. Array analyses of transcription in multiple murine prostate tumor models featuring PI3K/AKT pathway activation allowed construction of a pathway signature that may be useful in predicting the prognosis of human prostate tumors.

Background: Transcription factor Stat5a/b is highly critical for the viability of human prostate cancer cells in vitro and for prostate tumor growth in vivo. Stat5 is constitutively active in clinical prostate cancers but not in the normal human prostate epithelium. Moreover, Stat5a/b activation in prostate cancer is associated with high histological grade of prostate cancer. However, the molecular mechanisms underlying constitutive activation of Stat5a/b in prostate cancer are unclear. The receptor-associated tyrosine kinase Jak2 is a known key activator of Stat5a/b in prostate cancer cells in response to ligand stimulation. Recently, a single gain-of-function point mutation of JAK2 was described in myeloproliferative diseases leading to constitutive Jak2 kinase activity, subsequent Stat5a/b activation and involvement of V617F Jak2 in the pathogenesis of myeloproliferative disorders., Materials and Methods: We determined whether JAK2 undergoes the V617F activating mutation during clinical progression of human prostate cancer using a highly sensitive assay (amplification refractory mutation system) and a unique material of fresh specimens from organ-confined or castration-resistant prostate cancers., Results: The JAK2 V617F mutation was not found in any of the normal or malignant prostate samples analyzed in this study., Conclusions: Future work should focus on determining the molecular mechanisms other than V617F mutation of Jak2 resulting in continuous Stat5 activation in clinical prostate cancers.

Objective: To evaluate the involvement of thioredoxin reductase (TxnR), thioredoxin (Trx) and peroxiredoxins (Prdx) in prostate cancer (PCa) and to assess the potential prognostic importance of these redox-regulated pathways., Study Design: Expression of the isoforms TxnR2, Trx1 and Prdx2 was studied by immunohistochemistry on tissue microarrays (TMAs). In a prognostic TMA, 294 primary cases of PCa with a median follow-up of 49 months were stained for Trx1 and Prdx2. Another TMA containing benign prostatic tissue, atrophy, high grade prostatic intraepithelial neoplasia (HGPIN) and PCa from 40 patients was stained with all 3 antibodies. RES ULTS: All 3 proteins showed similar expression patterns, with the highest immunoreactivity in HGPIN followed by atrophy, PCa and benign tissue. TxnR2, Trx1 and Prdx2 were overexpressed in HGPIN and PCa compared with benign tissue (p < 0.001), and Trx1 and Prdx2 were also overexpressed in HGPIN compared with PCa (p < 0.001). Trx1 and Prdx2 did not correlate with biochemical recurrence., Conclusion: This study demonstrates up-regulation of the redox pathway proteins in PCa and its precursor lesions. The pathogenetic role of the redox system remains to be investigated.

Background: Inflammation has been strongly implicated in prostate carcinogenesis, but the precise molecular mechanisms linking inflammation and carcinogenic DNA damage are not known. Induction of the polyamine catabolic enzyme, spermine oxidase (SMO) has been linked to increased reactive oxygen species (ROS) and DNA damage in human gastric and lung epithelial cells and suggest direct mechanistic links between inflammation, SMO activity, ROS production, and epithelial carcinogenesis that are likely relevant in prostate cancer., Methods: Tissue microarrays consisting of matched normal and diseased specimens from patients diagnosed with prostate cancer, prostatic intraepithelial neoplasia (PIN), or proliferative inflammatory atrophy (PIA), as well as unaffected individuals, were stained for SMO expression and analyzed using image analysis techniques and TMAJ software tools., Results: Average SMO staining was significantly higher in prostate cancer and PIN tissues compared to patient-matched benign tissues. Benign tissues from prostate cancer, PIN, and PIA patients also exhibited significantly higher mean SMO expression versus tissues from prostate disease-free patients., Conclusions: Tissues from patients diagnosed with prostate cancer and PIN exhibit, on average, locally increased SMO expression in regions of prostatic disease and higher overall SMO expression in prostatic epithelial cells compared to healthy individuals. Further studies are warranted to directly examine the role of SMO-produced ROS in prostate carcinogenesis., ((c) 2008 Wiley-Liss, Inc.)

A significant proportion of human prostate cancers carry a chromosomal rearrangement resulting in the overexpression of the ETS transcription factor, ERG; however, the functional significance of this event is poorly understood. We report here that up-regulation of ERG transcript is sufficient for the initiation of prostate neoplasia. In agreement with measurements of ERG transcripts, we found that ERG protein is expressed in neoplastic human prostate epithelium. Overexpression of ERG in prostate cell lines increased cell invasion. Moreover, targeted expression of this transcript in vivo in luminal prostate epithelial cells of transgenic mice results in initiation of prostate neoplasia observed as the development of focal precancerous prostatic intraepithelial neoplasia (PIN). Similar to human cancers, luminal epithelial cells in these PIN lesions displace diminishing in numbers basal epithelial cells and establish direct contact with the stromal cell compartment. Loss of basal cells is considered to be one of the critical hallmarks of human prostate cancer; however, the mechanisms responsible for this event were unknown. We propose that up-regulation of ERG in human prostate cancer activates cell invasion programs that subsequently displace basal cells by neoplastic epithelium. Our data demonstrate that ERG plays an important causal role in the transformation of prostate epithelium and should be considered as a target for prevention or early therapeutic intervention.

Loss of the PTEN tumor suppressor is a common occurrence in human prostate cancer, particularly in advanced disease. In keeping with its role as a pivotal upstream regulator of the phosphatidylinositol 3-kinase signaling pathway, experimentally-induced deletion of Pten in the murine prostate invariably results in neoplasia. However, and unlike humans where prostate tumorigenesis likely evolves over decades, disease progression in the constitutively Pten deficient mouse prostate is relatively rapid, culminating in invasive cancer within several weeks post-puberty. Given that the prostate undergoes rapid androgen-dependent growth at puberty, and that Pten excisions during this time might be especially tumorigenic, we hypothesized that delaying prostate-specific Pten deletions until immediately after puberty might alter the pace of tumorigenesis. To this end we generated mice with a tamoxifen-inducible Cre recombinase transgene enabling temporal control over prostate-specific gene alterations. This line was then interbred with mice carrying floxed Pten alleles. Despite evidence of increased Akt/mTOR/S6K axis activity at early time points in Pten-deficient epithelial cells, excisions induced in the post-pubertal (6 wk-old) prostate yielded gradual acquisition of a range of lesions. These progressed from pre-malignant changes (nuclear atypia, focal hyperplasia) and low grade prostatic intraepithelial neoplasia (PIN) at 16-20 wks post-tamoxifen exposure, to overtly malignant lesions by approximately 1 yr of age, characterized by high-grade PIN and microinvasive carcinoma. In contrast, when Pten excisions were triggered in the pre-pubertal (2 week-old) prostate, neoplasia evolved over a more abbreviated time-frame, with a spectrum of premalignant lesions, as well as overt PIN and microinvasive carcinoma by 10-12 wks post-tamoxifen exposure. These results indicate that the developmental stage at which Pten deletions are induced dictates the pace of PIN development.

Objective: To investigate the correlation between the expressions of human telomerase reverse transcriptase (hTERT) and vascular endothelial growth factor (VEGF) in prostate cancer (PCa) and benign prostatic hyperplasia (BPH), and to determine if hTERT was correlated with VEGF-promoted tumor cell proliferation in prostate cancer., Materials and Methods: Immunohistochemistry was used to analyze the expressions of hTERT and VEGF in 60 cases of PCa and 60 cases of benign prostatic hyperplasia (BPH). Then their correlation in PCa was analyzed by Spearman correlative analysis., Results: The expressions of hTERT were detected in 38 cases of PCa and 10 cases of BPH. The expressions of VEGF were detected in 46 cases of PCa and 28 cases of BPH. The expressions of hTERT and VEGF in PCa were significantly higher than those in BPH (P < 0.05). As a result of correlation analysis, it was found that with an increase of the expression of VEGF, the expression of hTERT also increased in PCa. Significant correlation was observed between the expressions of hTERT and VEGF in PCa (r = 0.8333, P < 0.05). But there was no significant correlation between the expressions of hTERT and VEGF in BPH (r = 0.3156, P > 0.05)., Conclusions: All experiences above indicate that hTERT was one of the important proteins in the proliferation-promoting effect of VEGF on tumor cells in PCa.

Enhanced mesenchymal expression of FGF10 led to the formation of multifocal PIN or prostate cancer. Inhibition of epithelial FGFR1 signaling using DN FGFR1 led to reversal of the cancer phenotype. A subset of the FGF10-induced carcinoma was serially transplantable. Paracrine FGF10 led to an increase in epithelial androgen receptor and synergized with cell-autonomous activated AKT. Our observations indicate that stromal FGF10 expression may facilitate the multifocal histology observed in prostate adenocarcinoma and suggest the FGF10/FGFR1 axis as a potential therapeutic target in treating hormone-sensitive or refractory prostate cancer. We also show that transient exposure to a paracrine growth factor may be sufficient for the initiation of oncogenic transformation.

Background: Human tissue kallikrein-related peptidases (genes, KLKs; proteins, KLKs) are a subgroup of serine proteases present in a variety of tissues and biological fluids. A number of human tissue KLKs are established or candidate serologic biomarkers for prostate cancer. Human kallikrein-related peptidase 12 (KLK12, KLK12), recently identified in our laboratory, is a novel member of the KLK gene family. Here, we report generation of antibodies against the full-length recombinant KLK12 (classical form) and the immunohistological localization of this KLK in normal and malignant prostate tissues., Methods: The mature form of KLK12 cDNA was amplified using PCR and cloned into a plasmid vector for protein production in E. coli. Following identification by mass spectroscopy, recombinant KLK12 was purified and used as immunogen in rabbits. Anti- KLK12 antibody was used for immunostaining of paraffin-embedded sections of human prostate tissue. Immunoexpression of KLK12 in benign and malignant prostate tissue was evaluated using a prostate cancer tissue array., Results: Anti-KLK12 antibody showed a predominantly apical and membranous staining of the luminal cells of the normal prostate in contrast with the predominantly diffuse cytoplasmic staining observed in both prostatic intra-epithelial neoplasia and adenocarcinomas. This was occasionally associated with an intense granular supranuclear staining. More than 95% of the prostate cancers on the tissue microarray were KLK12 positive., Conclusion: Higher levels of KLK12 in malignant prostatic glands, and the shift in subcellular localization of KLK12 in prostate cancer observed in this study point to the potential role of this kallikrein during prostate carcinogenesis., (2007 Wiley-Liss, Inc)

Identification of the signalling cascades that are differentially activated during prostatic tumourigenesis is a crucial step in the search for future molecular targets in this disease. The stress-activated protein kinase (SAPK) signalling cascade culminates in the phosphorylation of the JNK and p38 mitogen-activated protein kinases (MAPKs). Recently, the upstream activators of these proteins, the MAPK kinases (MKKs), have been implicated as inhibitors of tumour progression in a variety of clinical and experimental tumour models. This study evaluates MKK4, MKK6 and MKK7 expression during prostate cancer progression in humans and in the transgenic adenocarcinoma of a mouse prostate (TRAMP) model of prostate tumourigenesis. Benign prostate, prostatic intraepithelial neoplasia (PIN) lesions and tumour tissues were collected from 37 TRAMP mice. Additionally, six tissue microarrays were constructed with tumours from a matched group of 102 men who underwent radical prostatectomy. Tissues from 20 patients with extensive high-grade prostatic intraepithelial neoplasia (HGPIN) were also analysed. For all samples, immunohistochemical staining for MKK4, MKK6 and MKK7 was scored in normal and neoplastic glands. Staining intensities of MKK4, MKK6 and MKK7 were significantly increased in HGPIN and prostate cancer compared to surrounding normal glands in both the TRAMP and human samples (p < 0.0001 for all markers). Increased levels of MKK4 or MKK7 correlated with higher pathological stage at prostatectomy (p = 0.01 and p = 0.04). Using multivariate analysis, there was no association between protein levels and time to biochemical recurrence in the human samples. The up-regulation of MKK4, MKK6 and MKK7 during prostate cancer progression in both TRAMP and human tissues highlights an important role for the SAPK signalling cascade in prostatic neoplasia. The finding that higher MKK4 and MKK7 expression is associated with higher-stage prostatic tumours underscores the dynamic regulation of these proteins during prostatic tumourigenesis., (Copyright (c) 2007 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Objective: To explore the nuclear chromatin phenotype, overall epigenetic mechanisms, chromatin remodelling enzymes and their role as diagnostic biomarkers in prostate lesions, using high-resolution computerized quantitative digital image analysis (DIA)., Materials and Methods: A tissue microarray (TMA) was constructed using paraffin wax-embedded prostatic tissues from 78 patients, containing 30 cores of benign prostatic hyperplasia (BPH), 10 of low-grade prostatic intraepithelial neoplasia (LGPIN), 38 of prostate adenocarcinoma, 20 of BPH tissue excised at 0.6-1 mm from LGPIN lesions, and 10 of BPH prostatic tissues obtained 0.6-1 mm from high-grade PIN (HGPIN) lesions. Chromatin phenotype was assessed on haematoxylin-stained sections using high-resolution texture analysis. For quantitative immunohistochemistry, antibodies raised against acetylated histone H3 lysine 9 (AcH3K9), 5'methylcytidine (5MeC) and the chromatin remodelling ATPase ISWI (SNF2H and SNF2L) were used. The immunodensity was measured using DIA to determine the epigenetic profile of the cases. At least 60 nuclei were measured from each case., Results: There were many statistically significant differences in staining intensity and nuclear distribution patterns in chromatin phenotype and immunostaining (p

The proliferative inflammatory atrophy (PIA) is considered as a possible precursor of prostate intraepithelial neoplasia (PIN) or prostate cancer (PCa). In this study we assessed quantitatively the expression of AMACR, p63, COX-2, GST and iNOS in serial paraffin-embedded tissue sections obtained after radical prostatectomy of PCa patients (n = 30). The applicability of these markers to distinguish PIA, PIN and PCa was evaluated. We also compared the immunohistochemical expression profiles of AMACR, COX-2 and GST in the luminal and basal cells in lesions of PIN arisen in PIA or PIA alone. Two different patterns of COX-2 expression according to the p63 status of the basal cells were found. This observation gives us grounds to hypothesize that the diverse COX-2 patterns resulted from an initial basal cell damage which subsequently propagated to its luminal secretory cells progeny.

Objective: To study the morphologic features and clinical significance of atypical small acinar proliferation (ASAP) suspicious but not diagnostic of cancer in prostatic biopsies., Methods: The slides of 11 cases of prostatic needle biopsies collected during a two-year period with the diagnosis of ASAP were reviewed. Immunohistochemical study for 34betaE12, p63 and P504S was performed on the archival paraffin sections., Results: All the 11 ASAP cases were characterized by the presence of a few compacted small acini in the prostatic stroma. Six cases had acini of less than three in number. The acini were round or slightly irregular in shape. The nuclei were enlarged, round or irregular, arranged in single layer and focally separated by broad interval. The nucleoli were usually prominent. Cytoplasm was amphophilic or pale and the lumen border was often well-defined. Basophilic mucus was also seen in some of the lumen. Immunohistochemical study for 34betaE12 and p63 was negative, while that for P504S was positive. In 4 of the 11 cases, the acini were more than three in number, round or slightly irregular, but without cytologic atypia. The nuclei were slightly enlarged with small or inconspicuous nucleoli. Immunohistochemical study for 34betaE12 and p63 was negative or at most focally positive. P504S staining was either negative or weakly positive. Second repeat biopsy was carried out in all cases, and 4 of them (36%) showed definite adenocarcinomatous changes. The positive cases were those with fewer acini but definite cytologic atypia in the initial biopsy., Conclusions: ASAP is a morphologic interpretation closely associated with prostatic adenocarcinoma. The histologic features are suspicious of but not diagnostic of cancer, due to insufficient criteria in terms of acinar number, cytologic or architectural abnormalities. The positive rate in subsequent repeat biopsy is higher than that for cases with usual nodular hyperplasia.

Human telomerase detected by in situ hybridization has been demonstrated to be a useful tool for the diagnosis of malignancy and has also been tested by reverse transcriptase-polymerase chain reaction in several tumors such as hepatic cell carcinoma, melanoma, colonic carcinoma, gastric carcinoma, biliary carcinoma, breast carcinoma, mesothelioma, lung carcinoma, female tract carcinoma, and prostatic carcinoma. A monoclonal antibody (clone Tel-24) that allows for the detection of human telomerase reverse transcriptase (hTERT) in paraffin blocks of archival material has recently been developed. Carcinomas of cervix, endometrium, and breast have been studied by this method, but its value in prostatic carcinoma has not been explored; for that reason, we studied benign and malignant prostatic lesions by immunohistochemistry using paraffin embedded tissue. The aim of the study was to define the sensitivity and specificity of hTERT in prostate cancer, in comparison with alpha-methylacyl-coenzyme A racemase (AMACR) (P504-S). Fifty-five specimens of diverse prostatic lesions were selected for study (43 needle biopsies and 12 transurethral resections); there were 61 malignancies (47 infiltrating carcinomas and 14 high-grade prostatic intraepithelial neoplasias [PIN]) and 29 benign lesions (10 basal cell hyperplasias, 12 nodular hyperplasias, 4 chronic prostatitis, and 3 atrophic glands). Signal for hTERT nucleolar was detected in 31 of 47 infiltrating adenocarcinomas, in 11 of 14 PIN, and in none of 27 benign lesions (sensitivity, 71%; specificity, 100%). Diffuse cytoplasmic positivity for AMACR was found in 37 of 41 infiltrating adenocarcinomas, in 7 of 7 PIN, and in 6 of 22 benign lesions (sensitivity, 91%; specificity, 72%). These results indicate that hTERT is highly specific of malignancy, with no false-positive cases; however, it had lower sensitivity than AMACR.

The tumor suppressor PTEN is frequently inactivated in human cancers. A major downstream effector of PTEN is Akt, which is hyperactivated via PTEN inactivation. It is not known, however, whether diminished Akt activity is sufficient to inhibit tumorigenesis initiated by Pten deficiency. Here we showed that the deficiency of Akt1 is sufficient to dramatically inhibit tumor development in Pten+/- mice. Akt1 deficiency had a profound effect on endometrium and prostate neoplasia, two types of human cancer, in which PTEN is frequently mutated, and also affected thyroid and adrenal medulla tumors and intestinal polyps. Even haplodeficiency of Akt1 was sufficient to markedly attenuate the development of high-grade prostate intraepithelial neoplasia (PIN) and endometrial carcinoma. These results have significant implications for cancer therapy.

Aims: To investigate the possibility of detecting small focal prostatic cancer by alpha-methylacyl CoA racemase (AMACR)/P504S immunohistochemistry on needle biopsy specimens that were previously interpreted as negative for carcinoma on routine haematoxylin and eosin (H&E)-stained sections., Methods: Prostate needle biopsy specimens (n = 793) previously interpreted as benign prostatic tissue by conventional morphology from 239 patients with prostatic cancer diagnosed in other biopsy cores taken at the same biopsy session were stained with the P504S monoclonal antibody. If a biopsy specimen stained positively, two pathologists independently reviewed the original corresponding H&E-stained sections to establish the malignant diagnosis., Results: Eighty-four of the 793 biopsy specimens showed AMACR immunoreactivity; nine of these (9/793, 1.1%) contained previously unrecognized small focal prostatic carcinoma (Gleason 6, N = 8; Gleason 8, N = 1). Six of nine (67%) carcinomas showed foamy/pseudohyperplastic (N = 3) or atrophic (N = 3) features. Additionally, five biopsy specimens (5/793, 0.6%) with positive AMACR staining that did not meet the criteria for prostatic cancer on the original H&E slides were considered to be atypia., Conclusions: In this study, we found a 1.1% false-negative rate for carcinoma on routine H&E-stained sections. AMACR immunohistochemical staining has shown the ability to improve detection of small focal prostatic carcinoma that could be missed by conventional histological examination.

Ornithine decarboxylase (ODC) is the key enzyme in the polyamine synthesis pathway and is overexpressed in a variety of cancers. We have performed a detailed immunostaining analysis of the expression of ODC in normal, benign prostatic hyperplasia (BPH), and cancerous prostate tissues. We conclude that ODC is overexpressed in both BPH and neoplastic tissues and that ODC overexpression appears to be an early event in prostate carcinogenesis. The extent of overexpression decreases as cancer progresses. Interestingly, ODC overexpression was also detected in patients who underwent androgen ablation therapy, suggesting ODC overexpression may contribute to the androgen-independent survival of prostate cancer cells. ODC is perinuclear localized in BPH samples but is diffusely cytoplasmic in cancer samples. Having shown ODC overexpression in human prostate cancer, we developed prostate-specific ODC transgenic mice to further investigate whether ODC overexpression alone is a causal factor in prostate carcinogenesis. RT-PCR and immunostaining confirmed that ODC was overexpressed in a subset of prostate epithelial cells. Although minor nucleoli enlargements in some tissues were detected, gross morphological changes were not observed in transgenic prostates. Therefore, overexpression of ODC alone in this subset of prostate epithelial cells is not sufficient to induce prostate carcinogenesis.

Aim: Studies using a monoclonal (P504S) and a polyclonal antibody (p-AMACR) to alpha-methylacyl-CoA racemase (AMACR) have shown variable expression in prostate cancer (PCa). The goal is to compare the sensitivity of both antibodies in PCa and evaluate their utility in the work-up of atypical prostate needle biopsies (NBXs)., Methods and Results: A tissue microarray (TMA) with 248 samples of benign prostate, high-grade prostatic intraepithelial neoplasia (HGPIN) and PCa samples, 20 NBXs with minute PCa and 32 NBXs with 'atypical' foci were stained with P504S and p-AMACR. Ninety percent of PCa (76/76 TMA, 16/20 NBXs) showed predominantly strong p-AMACR expression while 87% (65/69 TMA, 16/20 NBXs) showed variable P504S expression (sensitivity 90% versus 87%, P = 0.10). In HGPIN, P504S and p-AMACR were positive in 77% and 91% of samples, respectively. In the 'atypical' NBXs group, 53% were classified as PCa, 12% benign and 35% atypical, suspicious for PCa, after review of the basal marker. Of atypical, suspicious for PCa, P504S/p-AMACR helped convert the diagnosis to PCa in 5/11 (45%) cases, where, despite negative basal cell markers, morphology was less than optimal., Conclusions: Differences between P504S and p-AMACR appear marginal and clinically insignificant. AMACR is negative in a subset of unequivocal minute PCa with both antibodies. However, when utilized in proper context, AMACR may offer significant advantage in converting an 'atypical' diagnosis to PCa where morphology and basal markers are less than optimal in resolving the diagnosis.

Reductase activity of 17beta-hydroxysteroid oxidoreductase in biopsy specimens of prostatic cancer and benign hyperplasia, and prostatic intraepithelial neoplasia and serum concentrations of testosterone, 5alpha-dihydrotestosterone, 4-androstene-3,17-dione were compared in patients and healthy individuals. Reductase activities of 17beta-hydroxysteroid oxidoreductase in soluble fraction of prostatic biopsy specimens decreased in the following order: prostatic cancer>prostatic intraepithelial neoplasia>>benign prostatic hyperplasia. No differences in serum concentrations of testosterone, 5alpha-dihydrotestosterone, 4-androstene-3,17-dione between these three groups of patients were found, while the mean serum concentration of these androgens in patients with prostatic tumors did not surpass the threshold normal values for men. Hence, high reductase activity of 17beta-hydroxysteroid oxidoreductase can be associated with pathogenetic mechanisms of human malignant prostatic tumors.

Many human epithelial cancers, particularly those with a poor prognosis, express high levels of fatty acid synthase (FAS), a key metabolic enzyme linked to the synthesis of membrane phospholipids in cancer cells. In view of the recent finding that in the human prostate cancer cell line LNCaP, overexpression of FAS can be largely attributed to constitutive activation of the phosphatidylinositol-3 (PI3) kinase/Akt kinase pathway, the activation status of the Akt pathway, and whether this activation coincides with increased FAS expression, was examined in clinical prostate cancer tissues. Using well-preserved frozen prostatic needle biopsies and a sensitive Envision detection technique, S473-phosphorylated Akt (pAkt) was found in 11/23 low-grade prostatic intraepithelial neoplasia (PIN) lesions, in all (36/36) high-grade PINs, and in all (86/86) invasive carcinomas. Non-neoplastic tissues were negative. Interestingly, in low-grade PINs and low-grade carcinomas, pAkt was mainly cytoplasmic or membrane-bound and was associated with moderate elevation of FAS expression. In 24/36 high-grade PINs and 82/88 invasive carcinomas, pAkt was found at least partly in the nucleus. Greater nuclear pAkt staining, and higher FAS expression, correlated with a higher Gleason score. In the light of previous findings that pAkt plays a causative role in the overexpression of FAS in cancer cells in culture, these data strongly suggest that high-level expression of FAS in prostate cancer tissues is linked to phosphorylation and nuclear accumulation of Akt., (Copyright 2005 Pathological Society of Great Britain and Ireland)

Background: In the prostate, conversion of testosterone to dihydrotestosterone (DHT), by the enzymes 5alpha-reductase types 1 and 2 (5alphaR1, 5alphaR2) is required for normal growth and probably also for development of prostate cancer (PCa). Finasteride, a 5alphaR2 inhibitor, was shown to reduce the prevalence of PCa in the Prostate Cancer Prevention Trial. However, inhibition of both 5alphaR isoenzymes causes a greater decrease in serum DHT. The aim of this study was to assess differential expression of these enzymes at various stages of PCa development., Methods: Immunostaining for 5alphaR1 and 5alphaR2, using specific, well-validated antibodies, was evaluated in 26 benign prostatic hyperplasia (BPH) (16 for 5alphaR2), 53 primary PCa (21 for 5alphaR2), 18 prostatic intraepithelial neoplasia (PIN), 12 primary PCa treated with neoadjuvant androgen ablation, 15 locally recurrent PCa specimens, and 18 PCa metastases., Results: The mean area of moderate plus high intensity staining for 5alphaR1 increased from 4.8 +/- 2.8% of total epithelial area in BPH, to 18.9 +/- 5.7% in PIN, 17.0 +/- 3.2% in primary cancer, 38.0 +/- 7.3% in recurrent cancer, and 55.8 +/- 8.5% in PCa metastases. The mean staining area for 5alphaR2 decreased from 58.8 +/- 7.2% in BPH, to 21.1 +/- 5.5% in PIN and 34.8 +/- 6.7% in primary PCa. Staining for 5alphaR2 was increased in recurrent cancer and PCa metastases compared to primary PCa, at 58.7 +/- 5.2% and 69.2 +/- 8.7%, respectively., Conclusions: 5alphaR1 immunostaining is increased and 5alphaR2 immunostaining is decreased during development of PCa. In addition, there is increased expression of both 5alphaR isozymes in recurrent and metastatic cancers, suggesting that both isozymes may be important in the development and progression of PCa., ((c) 2004 Wiley-Liss, Inc.)

Over the last decade, epidemiological, experimental and clinical studies have implicated oxidative stress in the development and progression of prostate cancer. Oxidative stress may be linked to the effects of androgens, anti-oxidant systems and the pre-malignant condition, high-grade prostatic intraepithelial neoplasia. Cyclooxygenase-2 activity has been linked with prostate carcinogenesis. Evidence suggests that oxidative stress and cyclo-oxygenase-2 activity may be mechanistically linked. Agents such as anti-oxidants and cyclo-oxgenase-2 inhibitors may be of value in the chemoprevention of prostate cancer. The feasibility of intervention with such agents will depend on the development and validation of biomarkers for clinical trials, particularly markers of oxidative damage caused by reactive oxygen species (ROS). A greater understanding of the molecular events associated with oxidative stress will enhance the development of such biomarkers and should result in better strategies for the chemoprevention of prostate cancer.

Objectives: To perform the first evaluation of the effects of the 5-alpha-reductase inhibitor class of drugs on cancer histopathologic features at radical prostatectomy in a placebo-controlled multicenter trial., Methods: We analyzed prostatectomy slides in a blinded manner from 17 men treated with dutasteride, an inhibitor of types 1 and 2 isoenzymes of 5-alpha-reductase, and 18 men treated with placebo for 5 to 11 weeks before radical prostatectomy. The histopathologic features of benign epithelium, high-grade prostatic intraepithelial neoplasia, and cancer were recorded, and the treatment effect was scored. Digital imaging analysis was used to measure the stroma/epithelium ratio and epithelial height, as well as the nuclear area in cancer., Results: In benign epithelium, treatment caused distinctive cytoarchitectural changes of atrophy and a decrease in the epithelial height (P = 0.053). The peripheral zone showed the most marked response to treatment. In cancer tissue, the tumor volume was significantly lower in the dutasteride-treated men than in the placebo-treated men (mean 15% versus 24%, respectively, P = 0.025), the percentage of atrophic epithelium was increased (P = 0.041), and the stroma/gland ratio was doubled (P = 0.046). The treatment alteration effect score was doubled (P = 0.055) and did not correlate with any Gleason score changes., Conclusions: After short-term dutasteride treatment, benign epithelium showed involution and epithelial shrinkage, and prostate cancer tissue demonstrated a decrease in epithelium relative to stroma. These findings indicate that dutasteride induces significant phenotypic alterations in both the benign and the neoplastic prostate, supportive of a chemopreventive or chemoactive role.

The existing models of cancer progression assume that a linear sequence of genetic and epigenetic events occurs during this process. In this representation every new event (either loss of a tumor-suppressor, or activation of a proto-oncogene) makes cells even more malignant. The result is a "super" cell that can form metastases at the distant sites. Metastatic cells are believed to carry all genetic and epigenetic characteristics that are necessary for metastasis formation. Recently, we have shown that cell-surface protease hepsin causes disorganization of the basement membrane and promotes prostate cancer progression and metastasis. In human prostate cancer hepsin is upregulated in the precancerous lesions and this upregulation is maintained in the primary tumors. Remarkably and completely unexpected for a metastasis-promoting gene, hepsin is expressed at low levels in metastatic lesions and the message is completely absent in metastasis-derived prostate cancer cell lines. These results demonstrate that genes that play an important role in metastatic process may exercise their role only at the specific fragments of cancer progression pathway (for example, during initial invasion and tissue disorganization in the primary organ) and may have no role in metastatic lesions. Future treatment of cancer patients may rely heavily on monitoring of tumor progression, as treatment efficient in attenuation of initial tumor progression may be inefficient or even adverse at the advance stages of disease.

Alpha-methylacyl-CoA racemase (AMACR), also known as P504S, is a recently identified molecular marker for prostate cancer. The expression of AMACR/P504S has also been observed in high-grade prostatic intraepithelial neoplasia (PIN), a precursor lesion of prostate cancer. However, a detailed study focusing on the analysis of AMACR/P504S expression in high-grade PIN has not been performed. In this study, we analyzed AMACR/P504S expression by immunohistochemistry in 3954 prostatic ducts and acini with high-grade PIN from 140 prostatectomy specimens. AMACR/P504S immunoreactivity was measured as negative (0), weakly positive (+1), moderately positive (+2), and strongly positive (+3). AMACR/P504S immunoreactivity was detected in 90.0% (126/140) of high-grade PIN cases, although only 41.5% (1642/3954) of prostatic glands involved by PIN showed AMACR/P504S immunoreactivity. A significantly higher AMACR/P504S-positive rate (56.0%) was found in isolated high-grade PIN glands adjacent to cancer (distance less than 5 mm) compared with those away from cancer (distance more than 5 mm; 14%, P < 0.0001). High-grade PIN glands adjacent to cancer also showed a higher (P < 0.0004) AMACR/P504S intensity (1.62) than did those away from cancer (1.11). Our results suggest that PIN strongly positive for AMACR/P504S might be more closely associated with cancer than PIN negative or weakly positive for AMACR/P504S. This study provides additional evidence to link high-grade PIN as a precursor lesion to prostatic adenocarcinoma.

The metabolism of arachidonic acid by either the cyclooxygenase (COX) or lipoxygenase (LOX) pathway generates eicosanoids, which have been implicated in the pathogenesis of a variety of human diseases, including cancer. They are believed to play important roles in tumor promotion, progression, and metastasis. Involvement of LOXs expression and function in tumor growth and metastasis has been reported in human tumor cell lines. Expressions of 5- and 12-LOX in prostate cancer (PC) patients, prostatic intraepithelial neoplasia (PIN), benign prostatic hyperplasia (BPH), and normal prostate (NP) tissues were examined, as well as effects of their inhibitors on cell proliferation in 2 PC cell lines (PC3, DU-145). Expression of 5- and 12-LOX protein was detected by immunohistochemistry. Effects of LOX inhibitors on prostate cancer cell growth were examined by MTT assay, and Hoechst staining was used to determine whether or not the LOX inhibitors induce apoptosis. While 5- and 12-LOX expressions were slightly detected in BPH and NP tissues, marked expressions of 5- and 12-lipoxygenase were detected in PIN and PC tissues. The LOX inhibitors caused marked reduction of prostate cancer cells in a concentration- and time-dependent manner. The LOX inhibitors caused marked inhibition of PC cells through apoptosis. LOX is induced in prostate cancer, and our results suggest that LOX inhibitors may mediate potent antiproliferative effects against prostate cancer cells. Thus, LOX may become a new target in treatment of prostate cancer.

Purpose: To investigate the localization and distribution of cytochrome P450 CYP1B1 protein expression in patients diagnosed with prostate carcinoma compared to those with bladder carcinoma. To validate CYP1B1 as a molecular target for the development of selective cancer therapeutics for use in combination with radiation., Methods and Materials: Prostatectomy specimens (n = 33) of moderate Gleason grade (3 + 3 and 3 + 4) were analyzed immunohistochemically for CYP1B1 protein expression using a specific monoclonal antibody for the enzyme. The intensity of CYP1B1 staining was assessed both semiquantitatively using visual scoring and quantitatively by spectral imaging microscopy using reference spectra and compared with bladder carcinoma (n = 22)., Results: CYP1B1 protein expression was present in 75% of prostate carcinomas (n = 27) compared to 100% of bladder carcinomas (n = 22). In both cases, CYP1B1 protein expression was heterogeneous and localized in the cytoplasm of the tumor cells but absent from the surrounding stromal tissue. CYP1B1 was also detected in premalignant prostatic intraepithelial neoplasia (n = 2, 100%), as well as noncancerous tissues, including benign prostatic hyperplasia (n = 27, 82%), metaplastic prostatic urothelium (n = 8, 100%), and hyperplastic prostatic urothelium (n = 14, 100%). Higher CYP1B1 protein expression in bladder vs. prostate carcinoma was confirmed by their corresponding average normalized absorbances (+/- standard deviation), measured as 1.40 +/- 0.44 and 0.55 +/- 0.09, respectively. Overall CYP1B1 staining intensity in prostate carcinoma was similar to that in prostatic intraepithelial neoplasia, benign prostatic hyperplasia, and hyper-/metaplastic urothelial tissue. No CYP1B1 was detected in normal prostate tissue., Conclusions: CYP1B1 is overexpressed in prostate carcinoma at a high frequency and is also detectable in the associated premalignant and hyperplastic tissue, implicating a possible link with malignant progression and CYP1B1 as a suitable target for therapy. Spectral imaging microscopy has highlighted differences in CYP1B1 protein expression between different cancers.

Background: Ectopic expression of fibroblast growth factor receptor 1 (FGFR1) tyrosine kinase in epithelial cells is associated with progression of prostate cancer. Ectopic expression by transfection of FGFR1 in premalignant epithelial cells from nonmalignant Dunning tumors accelerated time-dependent progression of epithelial cells to malignancy. This study was designed to test the effect of chronic androgen-dependent ectopic activity of FGFR1 in the normal adult mouse epithelium by gene targeting., Materials and Methods: Constitutively active FGFR1 (caFGFR1) was targeted to prostate epithelial cells using the androgen-dependent probasin (PB) promoter. Prostate tissues of three strains were characterized over a period of 2 years by HE staining, immunohistochemical analyses for cytokeratin and alpha-actin, and rate of androgen-induced regeneration after castration., Results: Relative to wildtype littermates, transgenic mice showed increased overall size, hyperplasia in epithelial, and, to a lesser extent, stromal compartments and nuclear atypia in epithelial cells of the prostate with increasing age. Androgen-induced regeneration after castration was enhanced at day 3 by two-fold in mice expressing ectopic caFGFR1., Conclusions: The ectopic presence and chronic activation of FGFR1 in mouse prostate epithelial cells induces progressive prostate intraepithelial neoplasia. These results confirm results suggested by the transplantable Dunning tumor and cell culture models that, in contrast to homeostasis-promoting resident FGFR2, chronic ectopic FGFR1 kinase activity in the epithelium disrupts homeostasis between stroma and epithelium. Although insufficient alone, it may cooperate with other oncogenic changes to promote epithelial cells down the path to malignancy., (Copyright 2003 Wiley-Liss, Inc.)