Evidence of Heavy Methylation in the Galectin 3 Promoter in Early Stages of Prostate Adenocarcinoma: Development and Validation of a Methylated Marker for Early Diagnosis of Prostate Cancer

Galectins, soluble intracellular and extracellular β-galactoside-binding proteins, are known to be involved in the progression and metastasis of various cancers, including prostate adenocarcinoma, but the detailed mechanism of their biological roles remains elusive. In the prostate cancer cell lines PC-3 and DU-145, galectin 3 (gal3) is present at normal levels, whereas in LNCaP, its expression is silenced. In LNCaP, the gal3 promoter was heavily methylated, whereas PC-3 or DU-145 cells showed negligible or no methylation in the gal3 promoter indicating a negative correlation between gal3 promoter methylation and its expression. On immunohistochemical analysis of normal and tumor prostate tissues, gal3 was found expressed both in nucleus and cytoplasm of benign prostatic hyperplasia, high-grade prostatic intraepithelial neoplasia, and stage I. The expression of the gal3 was found drastically downregulated in advanced stages and, interestingly, mostly in the cytoplasm. On methylation analysis, the gal3 promoter in stage II prostate adenocarcinoma (PCa) was found heavily methylated, whereas in stages III and IV, it was only lightly methylated. However, in stage I PCa, both heavy and light methylations were observed in the gal3 promoter. In normal and benign prostatic hyperplasia tissues, the gal3 promoter was almost unmethylated. The differential cytosine methylation in the gal3 promoter in stages I to IV PCa enabled us to develop and validate a methylation-specific polymerase chain reaction-based sensitive assay specific for stages I and II PCa. These stages are considered the critical stages for successful intervention, thus underscoring the significance of this diagnostic assay.     

Characterization of Heterogeneous Prostate Tumors in Targeted Pten Knockout Mice

Previously, we generated a preclinical mouse prostate tumor model based on PSA-Cre driven inactivation of Pten. In this model homogeneous hyperplastic prostates (4-5m) developed at older age (>10m) into tumors. Here, we describe the molecular and histological characterization of the tumors in order to better understand the processes that are associated with prostate tumorigenesis in this targeted mouse Pten knockout model. The morphologies of the tumors that developed were very heterogeneous. Different histopathological growth patterns could be identified, including intraductal carcinoma (IDC), adenocarcinoma and undifferentiated carcinoma, all strongly positive for the epithelial cell marker Cytokeratin (CK), and carcinosarcomas, which were negative for CK. IDC pattern was already detected in prostates of 7–8 month old mice, indicating that it could be a precursor stage. At more than 10 months IDC and carcinosarcoma were most frequently observed. Gene expression profiling discriminated essentially two molecular subtypes, denoted tumor class 1 (TC1) and tumor class 2 (TC2). TC1 tumors were characterized by high expression of epithelial markers like Cytokeratin 8 and E-Cadherin whereas TC2 tumors showed high expression of mesenchyme/stroma markers such as Snail and Fibronectin. These molecular subtypes corresponded with histological growth patterns: where TC1 tumors mainly represented adenocarcinoma / intraductal carcinoma, in TC2 tumors carcinosarcoma was the dominant growth pattern. Further molecular characterization of the prostate tumors revealed an increased expression of genes associated with the inflammatory response. Moreover, functional markers for senescence, proliferation, angiogenesis and apoptosis were higher expressed in tumors compared to hyperplasia. The highest expression of proliferation and angiogenesis markers was detected in TC2 tumors. Our data clearly showed that in the genetically well-defined PSA-Cre;Pten-loxP/loxP prostate tumor model, histopathological, molecular and biological heterogeneity occurred during later stages of tumor development.     

Adiponectin as a potential marker of prostate cancer progression: studies in organ-confined and locally advanced prostate cancer

Serum levels of adiponectin were measured in patients with benign prostatic hyperplasia and prostate cancer of pT2 and pT3 stage. Adiponectin ELISA assay, immunohistochemistry, and selected metabolic and biochemical parameters measurement was performed in 25 patients with benign prostatic hyperplasia and 43 with prostate cancer (17 patients with organ-confined and 26 patients with locally advanced disease). Serum adiponectin levels did not differ between prostate benign hyperplasia and cancer clinical stage T2, but was significantly higher in pT3 relative to pT2 group (14.51+/-4.92 vs. 21.41+/-8.12, P = 0.003). Tissue immunohistochemistry showed enhanced staining in neoplastic prostate glands and intraepithelial neoplasia relative to benign prostatic hyperplasia without distinction between disease grade and stage. Serum adiponectin levels are higher in locally advanced relative to organ-confined prostate cancer and may thus serve as an auxiliary marker providing further improvement for discrimination between pT2 and pT3 stages.     

Transforming growth factor beta 1 and androgen receptors in prostate neoplasia

OBJECTIVE: To investigate the interplay between transforming growth factor (TGF) beta 1, androgen receptors and stromal-epithelial interactions in benign prostatic hyperplasia (BPH), prostate intraepithelial neoplasia (PIN) and prostate carcinoma areas of prostate neoplasia. STUDY DESIGN: In this immunohistochemical study we investigated staining patterns and then determined the correlation between TGF-beta 1 expression and androgen receptor status in the epithelium and stroma of 60 paraffin-embedded tissues from radical prostatectomies. RESULTS: Staining patterns differed in the epithelium and stroma of tumor and peritumor prostatic tissue. TGF-beta 1 immunostaining (H-scores) in the epithelium and stroma increased significantly from BPH to PIN and from BPH to prostate carcinoma in the epithelium (P < .05), whereas androgen receptor (AR) immunoreactivity significantly (P < .05) increased from BPH to PIN to prostatic carcinoma in epithelium and stroma. TGF-beta 1 did not correlate with histologic grade of differentiation, whereas AR proteins were more strongly expressed in Gleason score 5 and 6 than score 7 tumors (P < .05). Nonlinear regression showed a significant correlation (P < .01) between TGF-beta 1 and AR expression only in the stromal compartment of PIN. CONCLUSION: These findings argue in favor of an interaction between TGF-beta 1 and AR in the early stages of prostate carcinogenesis and suggest that TGF-beta 1 plays a central role in stromal-epithelial interactions during the early stages of malignant transformation.     

Dietary prevention of hormone refractory prostate cancer in Lobund-Wistar rats: a review of studies in a relevant animal model

Lobund-Wistar (LW) rats, which have high testosterone levels, are predisposed to develop hormone-refractory prostate cancer (HRPC) spontaneously and by methylnitrosourea (MNU) induction, and the development of HRPC progresses through 2 stages. This paper reviews several studies in which LW rats were placed on soy-containing diets and were evaluated for development of either spontaneous or MNU-induced prostate cancer. The premalignant, testosterone-dependent stage is inhibited by testosterone deprivation. In the absence of testosterone deprivation, tumorigenesis progresses spontaneously to the testosterone-independent refractory stage. In LW rats: moderate caloric restriction prevented development of spontaneous prostate cancer; dietary 4-hydroxyphenylretinamide prevented MNU-induced prostate cancer; and dietary supplementation with soy protein isolate with high isoflavones prevented spontaneous and induced tumors and led to moderate reduction of serum testosterone. In rats 12 mo of age and younger, changing from the control diet to the soy+isoflavone diet significantly prevented progression of spontaneous tumors to the refractory stage of disease. Tumors that developed spontaneously and after MNU induction showed similar developmental stages and morphology, but MNU-induced tumors had shorter latency periods before development. The accumulated data indicate that soy-based diets are effective in the prevention of prostate cancer.     

Stage-specific alterations of DNA methyltransferase expression, DNA hypermethylation, and DNA hypomethylation during prostate cancer progression in the transgenic adenocarcinoma of mouse prostate model

We analyzed DNA methyltransferase (Dnmt) protein expression and DNA methylation patterns during four progressive stages of prostate cancer in the transgenic adenocarcinoma of mouse prostate (TRAMP) model, including prostatic intraepithelial neoplasia, well-differentiated tumors, early poorly differentiated tumors, and late poorly differentiated tumors. Dnmt1, Dnmt3a, and Dnmt3b protein expression were increased in all stages; however, after normalization to cyclin A to account for cell cycle regulation, Dnmt proteins remained overexpressed in prostatic intraepithelial neoplasia and well-differentiated tumors, but not in poorly differentiated tumors. Restriction landmark genomic scanning analysis of locus-specific methylation revealed a high incidence of hypermethylation only in poorly differentiated (early and late) tumors. Several genes identified by restriction landmark genomic scanning showed hypermethylation of downstream regions correlating with mRNA overexpression, including p16INK4a, p19ARF, and Cacna1a. Parallel gene expression and DNA methylation analyses suggests that gene overexpression precedes downstream hypermethylation during prostate tumor progression. In contrast to gene hypermethylation, genomic DNA hypomethylation, including hypomethylation of repetitive elements and loss of genomic 5-methyldeoxycytidine, occurred in both early and late stages of prostate cancer. DNA hypermethylation and DNA hypomethylation did not correlate in TRAMP, and Dnmt protein expression did not correlate with either variable, with the exception of a borderline significant association between Dnmt1 expression and DNA hypermethylation. In summary, our data reveal the relative timing of and relationship between key alterations of the DNA methylation pathway occurring during prostate tumor progression in an in vivo model system.     

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.     

A PINK1-mediated mitophagy pathway decides the fate of tumors—to be benign or malignant?

Macroautophagy/autophagy plays a dual role in cancer depending on the stage of tumorigenesis. Autophagy prevents tumor initiation by suppressing chronic tissue damage, inflammation, accumulation of damaged organelles and genome instability. Autophagy can also sustain tumor metabolism and provide nutrients for tumor growth and survival via nutrient recycling. Moreover, autophagy is required for benign tumors to progress to malignant tumors. Emerging evidence indicates that autophagy or mitophagy can inactivate tumor suppressors such as TP53/TRP53/p53 to promote tumor progression once carcinogenesis has been initiated.     

Tumor models for prostate cancer exemplified by fibroblast growth factor 8-induced tumorigenesis and tumor progression

Prostate cancer is a very common malignancy among Western males. Although most tumors are indolent and grow slowly, some grow and metastasize aggressively. Because prostate cancer growth is usually androgen-dependent, androgen ablation offers a therapeutic option to treat post-resection tumor recurrence or primarily metastasized prostate cancer. However, patients often relapse after the primary response to androgen ablation therapy, and there is no effective cure for cases of castration-resistant prostate cancer (CRPC). The mechanisms of tumor growth in CRPC are poorly understood. Although the androgen receptors (ARs) remain functional in CRPC, other mechanisms are clearly activated (e.g., disturbed growth factor signaling). Results from our laboratory and others have shown that dysregulation of fibroblast growth factor (FGF) signaling, including FGF receptor 1 (FGFR1) activation and FGF8b overexpression, has an important role in prostate cancer growth and progression. Several experimental models have been developed for prostate tumorigenesis and various stages of tumor progression. These models include genetically engineered mice and rats, as well as induced tumors and xenografts in immunodeficient mice. The latter was created using parental and genetically modified cell lines. All of these models greatly helped to elucidate the roles of different genes in prostate carcinogenesis and tumor progression. Recently, patient-derived xenografts have been studied for possible use in testing individual, specific responses of tumor tissue to different treatment options. Feasible and functional CRPC models for drug responsiveness analysis and the development of effective therapies targeting the FGF signaling pathway and other pathways in prostate cancer are being actively investigated.     

Molecular alterations during insulinoma tumorigenesis

Insulinomas are the most common functioning endocrine pancreatic tumors (EPTs). They present with clinical symptoms as a consequence of hypoglycemia induced by inappropriate insulin secretion. The etiology of these tumors is poorly understood. Some tumors may harbor MEN1 gene mutations, the susceptibility gene of the multiple endocrine neoplasia type I syndrome, but most cases show wildtype MEN1. Currently, no reliable clinical tests are available to differentiate benign from malignant tumors. Approximately 30% of the tumors are unresectable, and they often show different growth rates, which hampers treatment. Therefore, a better understanding of the molecular processes underlying the development and progression of insulinomas is required to improve diagnosis, prognosis and therapy. Here we summarize the progress that has been made in insulinoma research in the past decade. We describe the clinical detection, classification and treatment of these tumors, and review the multiplicity of molecular and genetic studies that investigated tumor development and progression using either primary tumors, transgenic mouse models or tumor-derived cell lines. The identification of many interactors of the MEN1 gene product menin, as well as recurrent chromosomal abnormalities that pinpoint candidate genes of interest will likely result in a better understanding of the molecular pathways involved in insulinoma tumorigenesis. In addition, these studies will pave the way for the identification of novel targets for therapeutical intervention and more reliable markers for clinical diagnosis and prognosis.     

Dietary leucine improves whole-body insulin sensitivity independent of body fat in diet-induced obese Sprague–Dawley rats

Dairy foods and dietary calcium (Ca) are potential regulators of body weight and insulin sensitivity. The specific components of dairy responsible for these actions are not known but may include leucine. Our objective was to determine the effect of dietary protein (casein, skim milk or leucine) and Ca level [low, 0.67% (LC) or high, 2.4% (HC)] on adiposity and insulin sensitivity. Obesity was induced in Sprague–Dawley rats with a 6-week period of high-fat/high-sucrose (HFHS) diet intake. Rats were randomly assigned to one of six HFHS diets for 8 weeks where dietary protein was provided as casein, skim milk or casein enriched with leucine, and contained either LC or HC. Body composition via dual-energy x-ray absorptiometry and insulin sensitivity via euglycemic–hyperinsulinemic clamp were measured. Microarray was used to assess gene expression in liver and skeletal muscle. Rats fed leucine had greater insulin sensitivity than those fed casein or skim milk (P<.05). Dietary protein differentially regulated hepatic and skeletal muscle genes associated with insulin, peroxisome proliferator-activated receptor and mammalian target of rapamycin pathways. Specifically, two key genes responsible for insulin sensitivity, hepatic insulin receptor substrate (IRS) and protein kinase B (Akt), were altered in hepatic tissue in response to leucine. Rats fed skim milk and leucine diets had lower body weight compared to those fed casein (P<.05). HC reduced fat mass compared to LC (P<.05). While skim milk and leucine both reduced fat mass, only leucine improved insulin sensitivity compared to casein. Differential expression of genes such as IRS and Akt may be responsible for changes in insulin sensitivity in obese rats.     

Aberrant methylation of p16, HIC1, N33, and GSTP1 in tumor epithelium and tumor-associated cells in prostate cancer

The methylation status of p16, HIC1, N33, and GSTP1, which are involved in prostate carcinogenesis, was studied in prostate tissue samples containing neoplasms. Malignant acini, prostatic intraepithelial neoplasia (PIN) and benign prostatic hyperplasia (BPH) foci, and stroma surrounding glandular structures of each type were detected in histological sections, using laser capture microdissection of prostate tissue. High levels of methylation were found in tumor epithelium and adjacent tumor-associated stromal cells. Epigenetic changes in the stroma are indicative of a major role of tumor microenvironment in cancer development and progression. The methylation status of p16, HIC1, N33, and GSTP1 was also assessed in prostate biopsy material and operative tumor samples without laser capture microdissection. The methylation frequencies of all genes in tumor samples were considerably lower than those in microdissected tumor samples (HIC1, 71% vs. 89%; p16, 22% vs. 78%; GSTP1, 32% vs. 100%; and N33, 20% vs. 33%, respectively). It was concluded that laser capture micro-dissection is required in molecular analysis of tumors of this type.     

Lack of an Additive Effect between the Deletions of Klf5 and Nkx3-1 in Mouse Prostatic Tumorigenesis

Prostate cancer is one of the most common malignancies.The development and progression of prostate cancer are driven by a series of genetic and epigenetic events including gene amplification that activates oncogenes and chromosomal deletion that inactivates tumor suppressor genes.Whereas gene amplification occurs in human prostate cancer,gene deletion is more common,and a large number of chromosomal regions have been identified to have frequent deletion in prostate cancer,suggesting that tumor suppressor inactivation is more common than oncogene activation in prostatic carcinogenesis (Knuutila et al.,1998,1999;Dong,2001).Among the most frequently deleted chromosomal regions in prostate cancer,target genes such as NKX3-1 from 8p21,PTENfrom 10q23 andATBF1 from 16q22 have been identified by different approaches (He et al.,1997;Li et al.,1997;Sun et al.,2005),and deletion of these genes in mouse prostates has been demonstrated to induce and/or promote prostatic carcinogenesis.For example,knockout of Nkx3-1 in mice induces hyperplasia and dysplasia (Bhatia-Gaur et al.,1999;Abdulkadir et al.,2002) and promotes prostatic tumorigenesis (Abate-Shen et al.,2003),while knockout of Pten alone causes prostatic neoplasia (Wang et al.,2003).Therefore,gene deletion plays a causal role in prostatic carcinogenesis (Dong,2001).     

GnRH受体与TRAIL在前列腺肿瘤中表达的研究

目的研究促性腺激素释放激素受体(GnRH-R)和凋亡相关蛋白(TRAIL)在前列腺肿瘤中的表达及临床意义.方法采用SABC免疫组化法及原位定量法,对前列腺肿瘤和前列腺增生患者中GnRH-R和TRAIL的表达进行检测及半定量分析.结果在前列腺癌组织高分化的11例,中分化的4例以及低分化的5例中,各种不同分化的癌组织均显示不同程度的GnRH-R和TRAIL阳性免疫反应,原位定量显示GnRH-R与TRAIL的表达均分别随着组织分化程度增高而增高(P<0.05),并且TRAIL较GnRH-R阳性免疫反应强.结论 GnRH-R和TRAIL表达量可能与前列腺恶性肿瘤的发生与发展有关.     

Multistage carcinogenesis induced by ras and myc oncogenes in a reconstituted organ

ras and myc oncogenes were able to induce distinct phenotypic alterations, resembling different types of premalignant lesions, when introduced into approximately 0.1% of the cells used to reconstitute the mouse prostate gland. While ras induced dysplasia in combination with angiogenesis, myc induced a hyperplasia of the otherwise normally developed organ. ras and myc together induced primarily carcinomas. However, tumor progression was also associated with additional genetic alterations involving gene amplification. Our data indicate that specific types of benign premalignant lesions may reflect the activation of different single oncogenes, and that the consecutive activation of multiple oncogenes could be a causal event in the step-like progression of tumorigenesis.     

Beyond prostate-specific antigen: new serologic biomarkers for improved diagnosis and management of prostate cancer

The use of total prostate-specific antigen (tPSA) measurement has dramatically improved the ability to detect prostate cancer at earlier stages. However, as the number of men presenting with advanced disease (and high tPSA levels) has decreased, and given the fact that tPSA is highly reflective of benign prostatic hyperplasia, the need has emerged for novel biomarkers specifically associated with prostate cancer in order to improve predictive models. Several new biomarkers have shown promise, and studies continue to investigate the role of these markers in the detection, staging, and prognosis of prostate cancer. As new useful biomarkers continue to emerge, guidelines for their employment, as well as coordination of further research studies, are needed; a systematic, phased, nomogram-based model is a rational way to manage these efforts.     

Additive Effect of Zfhx3/Atbf1 and Pten Deletion on Mouse Prostatic Tumorigenesis

The phosphatase and tensin homolog(PTEN) and the zinc finger homeobox 3(ZFHX3)/AT-motif binding factor 1(ATBF1) genes have been established as tumor suppressor genes in prostate cancer by their frequent deletions and mutations in human prostate cancer and by the formation of mouse prostatic intraepithelial neoplasia(mPIN) or tumor by their deletions in mouse prostates.However,whether ZFHX3/ATBF1 deletion together with PTEN deletion facilitates prostatic tumorigenesis is unknown.In this study,we simultaneously deleted both genes in mouse prostatic epithelia and performed histological and molecular analyses.While deletion of one Pten allele alone caused low-grade(LG) mPIN as previously reported,concurrent deletion of 2fla3/Atbf1 promoted the progression to high-grade(HG)mPIN or early carcinoma.Zfhx3/Atbf1 and Pten deletions together increased cell proliferation,disrupted the smooth muscle layer between epithelium and stroma,and increased the number of apoptotic cells.Deletion of both genes also accelerated the activation of Akt and Erk1/2 oncoproteins.These results suggest an additive effect of ZFHX3/ATBF1 and PTEN deletions on the development and progression of prostate neoplasia.