Federica Rizzi

Demethylation of (Cytosine-5-C-methyl) DNA and regulation of transcription in the epigenetic pathways of cancer development

Cancer cells and tissues exhibit genome wide hypomethylation and regional hypermethylation. CpG-methylation of DNA (MeCpG-DNA) is defined as the formation of a C–C covalent bond between the 5′-C of cytosine and the –CH3 group of S-adenosylmethionine. Removal of the sole –CH3 group from the methylated cytosine of DNA is one of the many ways of DNA-demethylation, which contributes to activation of transcription. The mechanism of demethylation, the candidate enzyme(s) exhibiting direct demethylase activity and associated cofactors are not firmly established. Genome-wide hypomethylation can be obtained in several ways by inactivation of DNMT enzyme activity, including covalent trapping of DNMT by cytosine base analogues. Removal of methyl layer could also be occurred by excision of the 5-methyl cytosine base by DNA glycosylases. The importance of truly chemically defined direct demethylation of intact DNA in regulation of gene expression, development, cell differentiation and transformation are discussed in this contribution.

The clusterin paradigm in prostate and breast carcinogenesis

The biological functions of clusterin (CLU, also known as ApoJ, SGP2, TRPM-2, CLI) have been puzzling the researchers since its first discovery in the early 80s. We know that CLU is a single 9-exons gene expressing three protein forms with different sub-cellular localisations and diverse biological functions. Despite the many reports from many research teams on CLU action and its relation to tumourigenesis, contradictions in the data and alternative hypothesis still exist. Understanding the role of CLU in tumourigenesis is complicated not only by the existence of different protein forms but also by the changes of tumours over time and the selection pressures imposed by treatments such as hormone ablation or chemotherapy. This review focuses on recent discoveries concerning the role of CLU in prostate and breast cancer onset and progression. Although CLU acts primarily as a tumour suppressor in the early stages of carcinogenesis, consistent with its role in the involution of the prostate following castration, late stage cancer may overexpress CLU following chemotherapeutic drugs or hormonal ablation therapy. High expression of secreted or cytoplasmic CLU may represent a pro-survival stimulus because it confers increased resistance to killing by anti-cancer drugs or enhances tumour cell survival in specific niches.

A Novel Gene Signature for Molecular Diagnosis of Human Prostate Cancer by RT-qPCR

Background Prostate cancer (CaP) is one of the most relevant causes of cancer death in Western Countries. Although detection of CaP at early curable stage is highly desirable, actual screening methods present limitations and new molecular approaches are needed. Gene expression analysis increases our knowledge about the biology of CaP and may render novel molecular tools, but the identification of accurate biomarkers for reliable molecular diagnosis is a real challenge. We describe here the diagnostic power of a novel 8-genes signature: ornithine decarboxylase (ODC), ornithine decarboxylase antizyme (OAZ), adenosylmethionine decarboxylase (AdoMetDC), spermidine/spermine N(1)-acetyltransferase (SSAT), histone H3 (H3), growth arrest specific gene (GAS1), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and Clusterin (CLU) in tumour detection/classification of human CaP. Methodology/Principal Findings The 8-gene signature was detected by retrotranscription real-time quantitative PCR (RT-qPCR) in frozen prostate surgical specimens obtained from 41 patients diagnosed with CaP and recommended to undergo radical prostatectomy (RP). No therapy was given to patients at any time before RP. The bio-bank used for the study consisted of 66 specimens: 44 were benign-CaP paired from the same patient. Thirty-five were classified as benign and 31 as CaP after final pathological examination. Only molecular data were used for classification of specimens. The Nearest Neighbour (NN) classifier was used in order to discriminate CaP from benign tissue. Validation of final results was obtained with 10-fold crossvalidation procedure. CaP versus benign specimens were discriminated with (80±5)% accuracy, (81±6)% sensitivity and (78±7)% specificity. The method also correctly classified 71% of patients with Gleason score<7 versus ≥7, an important predictor of final outcome. Conclusions/Significance The method showed high sensitivity in a collection of specimens in which a significant portion of the total (13/31, equal to 42%) was considered CaP on the basis of having less than 15% of cancer cells. This result supports the notion of the “cancer field effect”, in which transformed cells extend beyond morphologically evident tumour. The molecular diagnosis method here described is objective and less subjected to human error. Although further confirmations are needed, this method posses the potential to enhance conventional diagnosis.

Anticancer Activity of Green Tea Polyphenols in Prostate Gland

Numerous evidences from prevention studies in humans, support the existence of an association between green tea polyphenols consumption and a reduced cancer risk. Prostate cancer is one of the most frequently diagnosed male neoplasia in the Western countries, which is in agreement with this gland being particularly vulnerable to oxidative stress processes, often associated with tumorigenesis. Tea polyphenols have been extensively studied in cell culture and animal models where they inhibited tumor onset and progression. Prostate cancer appears a suitable target for primary prevention care, since it grows slowly, before symptoms arise, thus offering a relatively long time period for therapeutic interventions. It is, in fact, usually diagnosed in men 50-year-old or older, when even a modest delay in progression of the disease could significantly improve the patients quality of life. Although epidemiological studies have not yet yielded conclusive results on the chemopreventive and anticancer effect of tea polyphenols, there is an increasing trend to employ these substances as conservative management for patients diagnosed with less advanced prostate cancer. Here, we intend to review the most recent observations relating tea polyphenols to human prostate cancer risk, in an attempt to outline better their potential employment for preventing prostate cancer.

Upregulation of clusterin in prostate and DNA damage in spermatozoa from bisphenol A-treated rats and formation of DNA adducts in cultured human prostatic cells.

Among endocrine disruptors, the xenoestrogen bisphenol A (BPA) deserves particular attention due to widespread human exposure. Besides hormonal effects, BPA has been suspected to be involved in breast and prostate carcinogenesis, which share similar estrogen-related mechanisms. We previously demonstrated that administration of BPA to female mice results in the formation of DNA adducts and proteome alterations in the mammary tissue. Here, we evaluated the ability of BPA, given with drinking water, to induce a variety of biomarker alterations in male Sprague-Dawley rats. In addition, we investigated the formation of DNA adducts in human prostate cell lines. In BPA-treated rats, no DNA damage occurred in surrogate cells including peripheral blood lymphocytes and bone marrow erythrocytes, where no increase of single-strand DNA breaks was detectable by comet assay and the frequency of micronucleated cells was unaffected by BPA. Liver cells were positive at transferase dUTP nick end labeling assay, which detects both single-strand and double-strand breaks and early stage apoptosis. BPA upregulated clusterin expression in atrophic prostate epithelial cells and induced lipid peroxidation and DNA fragmentation in spermatozoa. Significant levels of DNA adducts were formed in prostate cell lines treated either with high-dose BPA for 24 h or low-dose BPA for 2 months. The BPA-related increase of DNA adducts was more pronounced in PNT1a nontumorigenic epithelial cells than in PC3 metastatic carcinoma cells. On the whole, these experimental findings support mechanistically the hypothesis that BPA may play a role in prostate carcinogenesis and may, potentially, affect the quality of sperm.

Clusterin is a short half‐life, poly‐ubiquitinated protein, which controls the fate of prostate cancer cells

The Clusterin (CLU) gene produces different forms of protein products, which vary in their biological properties and distribution within the cell. Both the extra‐ and intracellular CLU forms regulate cell proliferation and apoptosis. Dis‐regulation of CLU expression occurs in many cancer types, including prostate cancer. The role that CLU plays in tumorigenesis is still unclear. We found that CLU over‐expression inhibited cell proliferation and induced apoptosis in prostate cancer cells. Here we show that depletion of CLU affects the growth of PC‐3 prostate cancer cells. Following siRNA targeting all CLU mRNA variants, all protein products quickly disappeared, inducing cell cycle progression and higher expression of specific proliferation markers (i.e., H3 mRNA, PCNA, and cyclins A, B1, and D) as detected by RT‐qPCR and Western blot. Quite surprisingly, we also found that the turnover of CLU protein is very rapid and tightly regulated by ubiquitin–proteasome mediated degradation. Inhibition of protein synthesis by cycloheximide showed that CLU half‐life is less than 2 h. CLU protein products were found poly‐ubiquitinated by co‐immuniprecipitation. Proteasome inhibition by MG132 caused stabilization and accumulation of all CLU protein products, including the nuclear form of CLU (nCLU), and committing cells to caspase‐dependent death. In conclusion, proteasome inhibition may induce prostate cancer cell death through accumulation of nCLU, a potential tumor suppressor factor. J. Cell. Physiol. 219: 314–323, 2009.

Genetic inactivation of ApoJ/clusterin: effects on prostate tumourigenesis and metastatic spread

ApoJ/Clusterin (CLU) is a heterodimeric protein localized in the nucleus, cytoplasm or secretory organelles and involved in cell survival and neoplastic transformation. Its function in human cancer is still highly controversial. In this study, we examined the prostate of mice in which CLU has been genetically inactivated. Surprisingly, we observed transformation of the prostate epithelium in the majority of CLU knockout mice. Either PIN (prostate intraepithelial neoplasia) or differentiated carcinoma was observed in 100 and 87% of mice with homozygous or heterozygous deletion of CLU, respectively. Crossing CLU knockout with TRAMP (prostate cancer prone) mice results in a strong enhancement of metastatic spread. Finally, CLU depletion causes tumourigenesis in female TRAMP mice, which are normally cancer free. Mechanistically, deletion of CLU induces activation of nuclear factor-kB, a potentially oncogenic transcription factor important for the proliferation and survival of prostate cells.

Polyphenon E®, a standardized green tea extract, induces endoplasmic reticulum stress, leading to death of immortalized PNT1a cells by anoikis and tumorigenic PC3 by necroptosis

Increasing doses of Polyphenon E®, a standardized green tea extract, were given to PNT1a and PC3 prostate epithelial cells mimicking initial and advanced stages of prostate cancer (PCa), respectively. Cell death occurred in both cell lines, with PNT1a being more sensitive [half-maximal inhibitory concentration (IC50) = 35 μg/ml] than PC3 (IC50 = 145 μg/ml) to Polyphenon E®. Cell cycle arrest occurred at G0/G1 checkpoint for PNT1a, and G2/M for PC3 cells. Endoplasmic reticulum stress (ERS) and unfolded protein response (UPR) occurred in both cell lines, with each exhibiting different timing in response to Polyphenon E®. Autophagy was transiently activated in PNT1a cells within 12 h after treatment as a survival response to overcome ERS; then activation of caspases and cleavage of poly (ADP ribose) polymerase 1 occurred, committing cells to anoikis death. Polyphenon E® induced severe ERS in PC3 cells, causing a dramatic enlargement of the ER; persistent activation of UPR produced strong upregulation of GADD153/CHOP, a key protein of ERS-mediated cell death. Thereafter, GADD153/CHOP activated Puma, a BH3-only protein, committing cells to necroptosis, a programmed caspase-independent mechanism of cell death. Our results provide a foundation for the identification of novel targets and strategies aimed at sensitizing apoptosis-resistant cells to alternative death pathways.

Clusterin (CLU) and Lung Cancer

Lung cancer is the leading cause of cancer-related mortality. It is categorized into two histological groups that have distinct clinical behaviors, the nonsmall cell lung cancers (NSCLC) and the small cell lung cancer (SCLC). When identified at an early stage, NSCLC is treated by surgical resection. However, patients who undergo surgical resection still have a relative low survival rate, primarily for tumor recurrence. Unfortunately, advances in cytotoxic therapy have reached a plateau and new approaches to treatment are needed together with new and better parameters for more accurate prediction of the outcome and more precise indication of the efficacy of the treatment. Several in vitro studies have examined the role of Clusterin (CLU) in carcinogenesis, lung cancer progression, and response to chemo- and radiotherapy. Studies performed in lung cancer cell lines and animal models showed that CLU is upregulated after exposure to chemo- and radiotherapy. A potential role proposed for the protein is cytoprotective. In vitro, CLU silencing by antisense oligonucleotides (ASO) and small-interfering RNAs (siRNA) directed against CLU mRNA in CLU-rich lung cancer cell lines sensitized cells to chemotherapy and radiotherapy and decreased their metastatic potential. In vivo, a recent work analyzed the prognostic role of CLU in NSCLC, showing that CLU-positive patients with lung cancer had a better overall survival and disease-free survival than those with CLU-negative tumors. These data are contradictory to the promising in vitro results. From the results of these studies we may hypothesize that in early-stage lung cancers CLU represents a positive biomarker correlating with better overall survival. In advanced patients, already treated with chemo- and radiotherapy, the induction of CLU may confer resistance to the treatments. However, many studies are needed to better understand the role of CLU in early-stage and advanced lung cancers with the aim to discriminate patients and specific local conditions that could benefit for a CLU knocking down treatment.

Clusterin (CLU) and Prostate Cancer

The role of clusterin (CLU) in prostate tumorigenesis is probably the most highly controversial, with evidence that CLU expression is increased or decreased in different cancer models. For example, some studies showed that CLU expression is increased in advanced stages of prostate cancer and that suppression of CLU expression sensitizes prostate cancer cells to chemotherapeutic drugs killing. In contrast with the hypothesis that CLU is a positive modulator of prostate cancer, we and others have observed that CLU is downregulated during human prostate cancer progression. Accordingly, a meta-analysis of available microarray data shows that CLU mRNA is significantly downregulated in prostate cancer tissue compared to normal prostate in 14 out of 15 independent studies. Recently, it was shown that CLU is silenced by promoter methylation in the murine TRAMP-C2 cell line, as well as in the human prostate cancer cell line LNCaP. Consistently, CLU expression was found to be significantly reduced in untreated and hormone-refractory human prostate carcinomas. This data suggest the importance of epigenetic events in the regulation of CLU in prostate cancer, supporting the idea that prostate cell transformation at early stages requires CLU silencing through chromatin remodeling.