Frequent 14-3-3 sigma promoter methylation in benign and malignant prostate lesions

14-3-3Sigma is a putative tumor suppressor gene involved in cell cycle regulation and apoptosis following DNA damage. 14-3-3Sigma loss of expression has been reported is several human cancers, including prostate adenocarcinoma and precursor lesions, and promoter hypermethylation has been proposed as the mechanism underlying gene silencing. Here, we investigate the frequency and extent of 14-3-3sigma promoter methylation in benign and cancerous prostate tissues. We examined tumor tissue from 121 patients with prostate carcinoma (PCa), 39 paired high-grade prostatic intraepithelial neoplasias (HGPIN), 29 patients with benign prostate hyperplasia (BPH), as well as four prostate cancer cell lines using quantitative methylation-specific PCR (QMSP). The percentage of methylated alleles (PMA) was calculated and correlated with clinical and pathological parameters. RT-PCR was performed in the cell lines to assess 14-3-3sigma mRNA expression. PCa, HGPIN, BPH, and cancer cell lines showed ubiquitous 14-3-3sigma promoter methylation. However, the PMA of HGPIN was significantly lower than that of PCa or BPH (P < 0.0001), while PCa and BPH did not significantly differ. The PMA did not correlate with any clinicopathological parameter. All prostate cancer cell lines expressed 14-3-3sigmamRNA. 14-3-3Sigma promoter methylation is a frequent event in prostate tissues and cancer cell lines. Furthermore, there is a progressive accumulation of neoplastic cells with 14-3-3sigma methylated alleles from HGPIN to PCa, suggesting a role for this epigenetic event in prostate carcinogenesis. However, other mechanisms besides promoter methylation might be required for effective 14-3-3sigma downregulation.     

Detection of pre-neoplastic and neoplastic prostate disease by MALDI profiling of urine

The heterogeneous progression to the development of prostate cancer (PCa) has precluded effective early detection screens. Existing prostate cancer screening paradigms have relatively poor specificity for cancer relative to other prostate diseases, commonly benign prostatic hyperplasia (BPH). A method for discrimination of BPH, HGPIN, and PCa urine proteome was developed through testing 407 patient samples using matrix assisted laser desorption-mass spectrometry time of flight (MALDI-TOF). Urine samples were adsorbed to reverse phase resin, washed, and the eluant spotted directly for MALDI-TOF analysis of peptides. The processing resolved over 130 verifiable signals of a mass range of 1000-5000 m/z to suggest 71.2% specificity and 67.4% sensitivity in discriminating PCa vs. BPH. Comparing BPH and HGPIN resulted in 73.6% specificity and 69.2% sensitivity. Comparing PCa and HGPIN resulted in 80.8% specificity and 81.0% sensitivity. The high throughput, low-cost assay method developed is amenable for large patient numbers required for supporting biomarker identification.     

Immunohistochemical staining of slit2 in primary and metastatic prostatic adenocarcinoma

BACKGROUND: Conflicting roles for Slit2, a protein involved in mediating the processes of cell migration and chemotactic response, have been previously described in prostate cancer. Here we use immunohistochemistry to evaluate the expression of Slit2 in normal donor prostate (NDP), benign prostatic hyperplasia (BPH), high-grade prostatic intraepithelial neoplasia (HGPIN), normal tissue adjacent to prostatic adenocarcinoma (NAC), primary prostatic adenocarcinoma (PCa), and metastatic prostatic adenocarcinoma (Mets). METHODS: Tissue microarrays were immunostained for Slit2. The staining intensities were quantified using automated image analysis software. The data was statistically analyzed using one-way analysis of variance with subsequent Tukey tests for multiple comparisons or a nonparametric equivalent. Eleven cases of NDP, 35 cases of NAC, 15 cases of BPH, 35 cases of HGPIN, 106 cases of PCa, and 37 cases of Mets were analyzed. RESULTS: Specimens of PCa and HGPIN had the highest absolute staining for Slit2. Significant differences were seen between PCa and NDP (P < .05), PCa and NAC (P < .05), HGPIN and NDP (P < .05), and HGPIN and NAC (P < .05). Whereas the average Mets staining was not significantly different from NDP or NAC, several individual Mets cases featured intense staining. CONCLUSIONS: To our knowledge, this represents the first study comparing the immunohistochemical profiles of Slit2 in PCa and Mets to specimens of HGPIN, BPH, NDP, and NAC. These findings suggest that Slit2 expression can be increased in HGPIN, PCa, and Mets, making it a potentially important biomarker for prostate cancer.     

15-lipoxygenase 2 (15-LOX2) is a functional tumor suppressor that regulates human prostate epithelial cell differentiation, senescence, and growth (size)

15-Lipoxygenase 2 (15-LOX2) is the major mammalian lipoxygenase expressed in normal human adult prostate and its expression is decreased or lost in high-grade prostate intraepithelial neoplasia (HGPIN) and prostate cancer (PCa). Our recent work has demonstrated that (1) 15-LOX2 has multiple alternatively spliced isoforms and is a negative cell-cycle regulator in normal human prostate (NHP) epithelial cells; (2) 15-LOX2 in NHP cells is positively regulated by Sp1 and negatively regulated by Sp3; (3) 15-LOX2 in NHP cells may be partially involved in cell differentiation; (4) 15-LOX2 is cell-autonomously upregulated in cultured NHP cells and its induction is associated with NHP cell senescence; and (5) 15-LOX2 is a functional prostate tumor suppressor. Here we summarize these new findings to provide a concise view of the potential biological functions of 15-LOX2 in NHP cells and of its deregulation in PCa development.     

Caveolin-1 upregulation contributes to c-Myc-induced high-grade prostatic intraepithelial neoplasia and prostate cancer

Previously we reported caveolin-1 (Cav-1) overexpression in prostate cancer cells and showed that it promotes prostate cancer progression. Here, we report that Cav-1 was overexpressed in 41.7% (15 of 36) of human high-grade prostatic intraepithelial neoplasia (HGPIN) specimens obtained during radical prostatectomies. Positive correlations exist between Cav-1-positive (Cav-1(+)) HGPIN and Cav-1(+) primary prostate cancer (rho = 0.655, P < 0.0001) and between Cav-1 and c-Myc expression in HGPIN (rho = 0.41, P = 0.032). To determine whether Cav-1 cooperates with c-Myc in development of premalignant lesions and prostate cancer in vivo, we generated transgenic mice with c-Myc overexpression driven by the ARR(2)PB promoter. In this ARR(2)PB-c-myc model, Cav-1 overexpression was found in mouse PIN (mPIN) lesions and prostate cancer cells and was associated with a significantly higher ratio of proliferative to apoptotic labeling in mPIN lesions than in the Cav-1-negative epithelia adjacent to those lesions (10.02 vs. 4.34; P = 0.007). Cav-1 overexpression was also associated with increased levels of P-Akt and VEGF-A, which were previously associated with Cav-1-induced prostate cancer cell survival and positive feedback regulation of cellular Cav-1 levels, respectively. In multiple prostate cancer cell lines, Cav-1 protein (but not mRNA) was induced by c-Myc transfection, whereas VEGF siRNA transfection abrogated c-Myc-induced Cav-1 overexpression, suggesting a c-Myc-VEGF-Cav-1 signaling axis. Overall, our results suggest that Cav-1 is associated with c-Myc in the development of HGPIN and prostate cancer. Furthermore, Cav-1 overexpression in HGPIN is potentially a biomarker for early identification of patients who tend to develop Cav-1(+) primary prostate cancer.     

Drug therapies for eradicating high-grade prostatic intraepithelial neoplasia in the prevention of prostate cancer

High-grade prostatic intraepithelial neoplasia (HGPIN) is a precursor to invasive prostate cancer observed as an isolated entity in a growing subset of men undergoing prostate biopsy. The presence of HGPIN predicts an increased risk of 1) coexisting occult prostate cancer at baseline and 2) delayed progression to prostate cancer. As such, men with HGPIN represent a population at high risk for the development of prostate cancer. Because the current recommended therapy is observation and delayed-interval biopsies until cancer develops, a well-tolerated therapeutic agent capable of interrupting the progression of HGPIN to cancer is highly desirable. Given the known cancer-stimulatory effects of estrogens in the prostate, the use of selective estrogen receptor modulators (SERMs) to provide an antiestrogen effect represents a novel strategy for prostate cancer prevention. Recent phase II data from trials using toremifene in the treatment of men with HGPIN validate the use of SERMs as a rational and provocative strategy for the prevention of prostate cancer.     

Survey of genetically engineered mouse models for prostate cancer: analyzing the molecular basis of prostate cancer development, progression, and metastasis

Genetically engineered mouse models have been generated to study the molecular basis of prostate cancer (PCa) development, progression, and metastasis. Selection of a prostate-specific promoter, such as the probasin (PB) and prostate specific antigen (PSA) promoters, is critical for generating sufficient levels of transgene expression to elicit a phenotypic response. To date, target genes have included growth factors, cell cycle regulators, pro- and anti-apoptotic proteins, steroid hormone and growth factor receptors, oncogenes, tumor suppressors, and homeobox genes. The experimental approaches used to generate these mouse models include overexpression of the transgene, knock-out/knock-in of transgene expression and conditional regulation of expression using Cre/lox technology. This review summarizes the promoters, which have been utilized to create genetically engineered mouse models for PCa. Furthermore, the effects of gene disruption on promoting low- and high-grade intraepithelial neoplasia (LGPIN and HGPIN, respectively), locally invasive carcinoma and metastatic lesions will be discussed. To date, the PB-Cre4 x PTENloxp/loxp model appears to be the only model that represents the entire continuum of prostate adenocarcinoma development, tumor progression, and metastasis, although models that develop prostatic neuroendocrine (NE) cancer can be generated by disrupting one genetic event. Indeed, analysis of bigenic mouse models indicates that two genetic events are generally required for progression from HGPIN to locally invasive adenocarcinoma and that two to five genetic events can promote metastasis to distant sites. Studying the effects of genetic perturbation on PCa biology will increase our understanding of the disease process and potentially provide targets for developing novel therapeutic approaches.     

Ki-67 immunolabeling in pre-malignant lesions and carcinoma of the prostate. Histological correlation and prognostic evaluation

The antigen Ki-67, which is associated with cell proliferation, has been demonstrated to be useful in predicting the development of human tumors. The objective of this study was to evaluate the prognostic utility of this biomarker in pre-malignant and malignant lesions of the prostate. A total of 162 prostate biopsies taken from patients diagnosed for benign prostatic hyperplasia (BPH, n=49), low grade prostatic intraepithelial neoplasia (LGPIN, n=53), high grade prostatic intraepithelial neoplasia (HGPIN, n=25) and carcinoma (CAR, n=35), were studied. Immunohistochemistry for Ki-67 was carried out on all the samples and the number of labeled cells was semi-quantitatively evaluated (weak, moderate or intense). In the non-invasive lesions, the presence of Ki-67-positive cells in the luminal layer of the epithelium was evaluated qualitatively as positive or negative. The correlation between the immunolabeling for Ki-67 and the histological diagnosis showed highly significant differences between BPH and CAR, LGPIN and CAR and HGPIN and CAR, with no significant differences being found among the other groups. Analysis of the immunolabeling in luminal cells of non-invasive lesions showed an increase in accordance with the increase in the degree of histological lesion, the greatest percentage being obtained in the HGPIN lesions (88.0%), with significant differences among all the groups. Bearing in mind that Ki-67 is a prognostic biomarker for cell proliferation, our results demonstrating the immunolabeling of Ki-67 in the luminal compartment of non-invasive lesions having the potential to evolve to malignancy, may have prognostic implications.     

Epithelium segmentation and automated Gleason grading of prostate cancer via deep learning in label‐free multiphoton microscopic images

In the current clinical care practice, Gleason grading system is one of the most powerful prognostic predictors for prostate cancer (PCa). The grading system is based on the architectural pattern of cancerous epithelium in histological images. However, the standard procedure of histological examination often involves complicated tissue fixation and staining, which are time‐consuming and may delay the diagnosis and surgery. In this study, label‐free multiphoton microscopy (MPM) was used to acquire subcellular‐resolution images of unstained prostate tissues. Then, a deep learning architecture (U‐net) was introduced for epithelium segmentation of prostate tissues in MPM images. The obtained segmentation results were then merged with the original MPM images to train a classification network (AlexNet) for automated Gleason grading. The developed method achieved an overall pixel accuracy of 92.3% with a mean F1 score of 0.839 for epithelium segmentation. By merging the segmentation results with the MPM images, the accuracy of Gleason grading was improved from 72.42% to 81.13% in hold‐out test set. Our results suggest that MPM in combination with deep learning holds the potential to be used as a fast and powerful clinical tool for PCa diagnosis.     

Androgen receptor signaling is required for androgen-sensitive human prostate cancer cell proliferation and survival

Background  

Androgens and androgen receptors (AR) regulate normal prostate development and growth. They also are involved in pathological development of prostatic diseases, including benign prostatic hyperplasia (BPH) and prostate cancer (PCa). Antiandrogen therapy for PCa, in conjunction with chemical or surgical castration, offers initial positive responses and leads to massive prostate cell death. However, cancer cells later appear as androgen-independent PCa. To investigate the role of AR in prostate cell proliferation and survival, we introduced a vector-based small interfering RNA (siRNA). This siRNA targeted 5'-untranslated region of AR mRNA for extended suppression of AR expression in androgen-sensitive human prostate LNCaP cells.     

Role of stromal tenascin-C in mouse prostatic development and epithelial cell differentiation

Deregulation of epithelial-stromal interactions is considered to play a critical role in the initiation and promotion of benign prostatic hyperplasia (BPH) and prostate carcinoma (PCa). Expression of tenascin-C (TN-C), an extracellular matrix (ECM) glycoprotein, is reportedly higher in BPH and PCa as compared with normal prostate. Remodeling of the ECM alters the homeostatic balance between epithelium and stroma, resulting in physiological changes in cellular functions. To investigate the role of TN-C in prostatic development and differentiation, we evaluated the morphological phenotype of TN-C knockout (KO) mouse prostate (ventral: VP, dorsolateral: DLP, and anterior: AP) and examined tissue recombinants composed of adult mouse DLP epithelium and fetal TN-C KO urogenital sinus mesenchyme (UGM). Histological analysis showed epithelial cell clusters protruding into the ductal lumens in TN-C KO AP and DLP. Interestingly, binucleated cells appeared in epithelium of TN-C KO DLP at 8 weeks. Simultaneously, androgen receptor (AR)-positive cells were decreased in TN-C KO epithelia. Similar to the TN-C KO phenotype, protruded epithelial clusters, binucleated cells, and AR-negative nuclei were induced in DLP epithelium by recombining with TN-C KO UGM. Our results suggest that stromal TN-C might be involved in maintaining epithelial cytodifferentiation, morphogenesis, and androgen receptor expression of normal prostate glands in adult mice.     

Metabolomic characterization of human prostate cancer bone metastases reveals increased levels of cholesterol

Background

Metastasis to the bone is one clinically important features of prostate cancer (PCa). Current diagnostic methods cannot predict metastatic PCa at a curable stage of the disease. Identification of metabolic pathways involved in the growth of bone metastases therefore has the potential to improve PCa prognostication as well as therapy.

Methodology/Principal Findings

Metabolomics was applied for the study of PCa bone metastases (n = 20) in comparison with corresponding normal bone (n = 14), and furthermore of malignant (n = 13) and benign (n = 17) prostate tissue and corresponding plasma samples obtained from patients with (n = 15) and without (n = 13) diagnosed metastases and from men with benign prostate disease (n = 30). This was done using gas chromatography-mass spectrometry for sample characterization, and chemometric bioinformatics for data analysis. Results were verified in a separate test set including metastatic and normal bone tissue from patients with other cancers (n = 7). Significant differences were found between PCa bone metastases, bone metastases of other cancers, and normal bone. Furthermore, we identified metabolites in primary tumor tissue and in plasma which were significantly associated with metastatic disease. Among the metabolites in PCa bone metastases especially cholesterol was noted. In a test set the mean cholesterol level in PCa bone metastases was 127.30 mg/g as compared to 81.06 and 35.85 mg/g in bone metastases of different origin and normal bone, respectively (P = 0.0002 and 0.001). Immunohistochemical staining of PCa bone metastases showed intense staining of the low density lipoprotein receptor and variable levels of the scavenger receptor class B type 1 and 3-hydroxy-3-methylglutaryl-coenzyme reductase in tumor epithelial cells, indicating possibilities for influx and de novo synthesis of cholesterol.

Conclusions/Significance

We have identified metabolites associated with PCa metastasis and specifically identified high levels of cholesterol in PCa bone metastases. Based on our findings and the previous literature, this makes cholesterol a possible therapeutic target for advanced PCa.     

Prostatic intraepithelial neoplasia: an overview

Prostatic intraepithelial neoplasia (PIN) is the most established precursor of prostatic carcinoma. The presence of prominent nucleoli within an existing duct structure is an easy way to identify the disorder. Four main patterns of high-grade PIN (HGPIN) have been described: tufting, micropapillary, cribriform, and flat. In addition to exhibiting similar cytologic features, both HGPIN and prostatic carcinoma are associated with increased incidence and severity with age, and with high rates of occurrence in the peripheral zone of the prostate. HGPIN and prostate cancer share genetic and molecular markers as well, with PIN representing an intermediate stage between benign epithelium and invasive malignant carcinoma. The clinical significance of HGPIN is that it identifies patients at risk for malignancy. With the increased use of extended biopsy protocols, clinicians are more likely to identify HGPIN and less likely to miss concurrent carcinoma. Androgen deprivation therapy decreases the prevalence and extent of PIN, and may play a role in chemoprevention. Preliminary studies suggest that selective estrogen receptor modulators may also prevent the progression of HGPIN to prostate cancer.     

Localization of annexins I, II, IV and VII in whole prostate sections from radical prostatectomy patients

Annexins (ANXs) represent a family of calcium and phospholipid binding proteins that are involved in several physiological processes e.g. signal transduction, cellular differentiation and proliferation. Since they are known to be dysregulated in a variety of cancers we investigated the immunolocalization of ANXs in whole prostate sections containing benign prostatic epithelium (BPE), benign prostatic hyperplasia (BPH), prostatic intraepithelial neoplasia (PIN) and prostate cancer (PCa) in order to evaluate their possible role during tumorigenesis. Samples were obtained from 28 patients undergoing radical prostatectomy. Gross sections of whole prostates were examined immunohistochemically for the distribution of ANX I, II, IV and VII. In BPE all ANXs were localized to the cell membranes and the cytoplasm of all gland cells. In BPH the immunoreactivity of ANX I and II was restricted to the basal cells of glands and expression pattern of ANX IV and VII was similar to BPE. In PIN only basal cells expressed ANX II. In PCa ANX II immunoreactivity was absent and weak ANX I and ANX IV immunoreactivity was restricted to the cytoplasm of tumor cells. ANX VII immunoreactivity was seen in some but not all tumor cells. Since ANX IV and VII expression did not show significant changes in PCa compared to non-neoplastic tissue and PIN an essential role during prostate tumourigenesis seems unlikely. In contrast, as progression from PIN to PCa is characterized by a reduction of ANX I and II this suggests that downregulation of these proteins could represent an important event in prostate carcinogenesis.     

Genome-wide differentially methylated genes in prostate cancer tissues from African-American and Caucasian men

Increasing evidence suggests that aberrant DNA methylation changes may contribute to prostate cancer (PCa) ethnic disparity. To comprehensively identify DNA methylation alterations in PCa disparity, we used the Illumina 450K methylation platform to interrogate the methylation status of 485,577 CpG sites focusing on gene-associated regions of the human genome. Genomic DNA from African-American (AA; 7 normal and 3 cancers) and Caucasian (Cau; 8 normal and 3 cancers) was used in the analysis. Hierarchical clustering analysis identified probe-sets unique to AA and Cau samples, as well as common to both. We selected 25 promoter-associated novel CpG sites most differentially methylated by race (fold change > 1.5-fold; adjusted P < 0.05) and compared the β-value of these sites provided by the Illumina, Inc. array with quantitative methylation obtained by pyrosequencing in 7 prostate cell lines. We found very good concordance of the methylation levels between β-value and pyrosequencing. Gene expression analysis using qRT-PCR in a subset of 8 genes after treatment with 5-aza-2′-deoxycytidine and/or trichostatin showed up-regulation of gene expression in PCa cells. Quantitative analysis of 4 genes, SNRPN, SHANK2, MST1R, and ABCG5, in matched normal and PCa tissues derived from AA and Cau PCa patients demonstrated differential promoter methylation and concomitant differences in mRNA expression in prostate tissues from AA vs. Cau. Regression analysis in normal and PCa tissues as a function of race showed significantly higher methylation prevalence for SNRPN (P = 0.012), MST1R (P = 0.038), and ABCG5 (P < 0.0002) for AA vs. Cau samples. We selected the ABCG5 and SNRPN genes and verified their biological functions by Western blot analysis and siRNA gene knockout effects on cell proliferation and invasion in 4 PCa cell lines (2 AA and 2 Cau patients-derived lines). Knockdown of either ABCG5 or SNRPN resulted in a significant decrease in both invasion and proliferation in Cau PCa cell lines but we did not observe these remarkable loss-of-function effects in AA PCa cell lines. Our study demonstrates how differential genome-wide DNA methylation levels influence gene expression and biological functions in AA and Cau PCa.     

Down-regulation of human DAB2IP gene expression mediated by polycomb Ezh2 complex and histone deacetylase in prostate cancer

Human DAB2IP (hDAB2IP), a novel GTPase-activating protein modulating the Ras-mediated signaling and tumor necrosis factor-mediated apoptosis, is a potent growth inhibitor in human prostate cancer (PCa). Loss of hDAB2IP expression in PCa is due to altered epigenetic regulation (i.e. DNA methylation and histone modification) of its promoter region. The elevated polycomb Ezh2, a histone methyltransferase, has been associated with PCa progression. In this study, we have demonstrated that an increased Ezh2 expression in normal prostatic epithelial cells can suppress hDAB2IP gene expression. In contrast, knocking down the endogenous Ezh2 levels in PCa by a specific small interfering RNA can increase hDAB2IP expression. The association of Ezh2 complex (including Eed and Suz12) with hDAB2IP gene promoter is also detected in PCa cells but not in normal prostatic epithelial cells. Increased Ezh2 expression in normal prostatic epithelial cells by cDNA transfection facilitates the recruitment of other components of Ezh2 complex to the hDAB2IP promoter region accompanied with the increased levels of methyl histone H3 (H3) and histone deacetylase (HDAC1). Consistently, data from PCa cells transfected with Ezh2 small interfering RNA demonstrated that reduced Ezh2 levels resulted in the dissociation of Ezh2 complex accompanied with decreased levels of both methyl H3 and HDAC1 from hDAB2IP gene promoter. We further unveiled that the methylation status of Lys-27 but not Lys-9 of H3 in hDAB2IP promoter region is consistent with the hDAB2IP levels in both normal prostatic epithelial cells and PCa cells. Together, we conclude that hDAB2IP gene is a target gene of Ezh2 in prostatic epithelium, which provides an underlying mechanism of the down-regulation of hDAB2IP gene in PCa.     

Tumor-suppressive functions of 15-Lipoxygenase-2 and RB1CC1 in prostate cancer

15-Lipoxygenase-2 (15-LOX2) is a human-specific lipid-peroxidizing enzyme most prominently expressed in epithelial cells of normal human prostate but downregulated or completely lost in > 70% of prostate cancer (PCa) cases. Transgenic expression of 15-LOX2 in the mouse prostate surprisingly causes hyperplasia. Here we first provide evidence that 15-LOX2-induced prostatic hyperplasia does not progress to PCa even in p53^+/− or p53^−/− background. More important, by generating 15-LOX2; Hi-Myc double transgenic (dTg) mice, we show that 15-LOX2 expression inhibits Myc-induced PCa development, such that in the 3-month- and 6-month-old dTg mice, there is a significant reduction in prostate intraneoplasia (PIN) and PCa prevalent in age-matched Hi-Myc prostates. The dTg prostates show increased cell senescence and expression of several senescence-associated molecules, including p27, phosphorylated Rb, and Rb1cc1. We further show that in HPCa, 15-LOX2 and c-Myc manifest reciprocal protein expression patterns. Moreover, RB1CC1 accumulates in senescing normal human prostate (NHP) cells, and in both NHP and RWPE-1 cells, the 15-LOX2 metabolic products 15(S)-HPETE and 15(S)-HETE induce RB1CC1. We finally show that unlike 15-LOX2, RB1CC1 is not lost but rather frequently overexpressed in PCa samples. RB1CC1 knockdown in PC3 cells enhances clonal growth in vitro and tumor growth in vivo. Together, our present studies provide evidence for tumor-suppressive functions for both 15-LOX2 and RB1CC1.