cytidylyl-3--5--guanosine and Prostatic-Neoplasms

XIAP-associated factor 1 (XAF1) is a new candidate tumor suppressor, which has been known to exert proapoptotic effects by interfering with the caspase-inhibiting activity of XIAP. To explore the XAF1's candidacy for a suppressor in urogenital tumorigenesis, we investigated the XAF1 status in a series of cancer cell lines and primary tumors derived from the bladder, kidney and prostate. Expression of XAF1 transcript was undetectable or extremely low in 60% (3/5) of bladder, 66% (10/15) of kidney, and 100% (3/3) prostate cancer cell lines. Abnormal reduction of XAF1 was also found in 33% (18/55) of primary bladder and 40% (8/20) of primary kidney tumors, and showed a correlation with advanced stage and high grade of bladder tumor. Hypermethylation at 14 CpG sites in the 5' proximal region of the XAF1 promoter was highly prevalent in cancers versus adjacent normal or benign tissues and tightly associated with reduced gene expression. XAF1 expression enhanced the apoptotic response of tumor cells to chemotherapeutic agents, such as etoposide or 5-FU. While XAF1 expression did not influence the subcellular distribution or expression of XIAP, it elevated the protein stability of p53 and its target gene expression. Moreover, the apoptosis-sensitizing and growth suppression function of XAF1 was markedly impeded by blockade of p53 function. Collectively, our study demonstrates that epigenetic alteration of XAF1 is frequent in human urogenital cancers and may contribute to the malignant progression of tumors by rendering tumor cells a survival advantage partially through the attenuated p53 response to apoptotic stresses.

Topics: Adaptor Proteins, Signal Transducing; Apoptosis Regulatory Proteins; Dinucleoside Phosphates; DNA Methylation; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Intracellular Signaling Peptides and Proteins; Kidney Neoplasms; Male; Neoplasm Proteins; Promoter Regions, Genetic; Prostatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; Tumor Suppressor Protein p53; Urinary Bladder Neoplasms; Urogenital Neoplasms

Recent studies have reported that the majority of prostate cancers express fusion genes in which the 5' region of the androgen-regulated TMPRSS2 gene is fused to an ETS family transcription factor, most commonly the ERG gene. We have characterized in detail the expression of TMPRSS2/ERG fusion mRNAs and correlated the isoforms expressed and expression levels with clinical outcome in cancers from men undergoing radical prostatectomy. Overall, 59% of clinically localized prostate cancers express the TMPRSS2/ERG fusion gene, confirming the initial observations of high frequency expression of this fusion mRNA in prostate cancer. There was significant variation in the alternatively spliced isoforms expressed in different cancers. Expression of an isoform, in which the native ATG in exon 2 of the TMPRSS2 gene is in frame with exon 4 of the ERG gene, was associated with clinical and pathologic variables of aggressive disease. Expression of other isoforms, in which the native ERG ATG in exon 3 was the first in-frame ATG, was associated with seminal vesicle invasion, which is correlated with poor outcome following radical prostatectomy. Cancers not expressing these isoforms tended to express higher levels of fusion mRNAs, and in this group, higher expression levels of fusion mRNA were present in cancers with early prostate-specific antigen recurrence. Thus, both the isoforms of TMPRSS2/ERG fusions expressed and expression level may affect prostate cancer progression.

Topics: Cell Line, Tumor; Dinucleoside Phosphates; Genetic Variation; Humans; Male; Prostatic Neoplasms; Recombinant Fusion Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Serine Endopeptidases

Estrogen receptor (ER)-beta is thought to exert anti-proliferative effects in the normal prostate but supports prostate cancer (PCa) cell survival. We previously reported that the receptor's expression declined as PCa developed in the gland but reappeared in lymph node and bone metastases. To investigate whether hypermethylation was the underlying mechanism for these phenomena, we first identified two CpG islands (CGIs) encompassing 41 CpG dinucleotides, located separately in the untranslated exon 0N and the promoter region of ER-beta. Using immunostained, laser capture-microdissected samples from 56 clinical specimens, we demonstrated an inverse relationship exists between the extent of ER-beta CGI methylation and receptor expression in normal, hyperplastic, premalignant, and malignant foci of the prostate and in lymph node and bone metastases. Treatment of PCa cell lines (LNCaP and DU145), that express little ER-beta mRNA, with a demethylating agent increased levels of receptor expression thus corroborating our in vivo findings that methylation is involved in ER-beta silencing. Methylation centers in the promoter region and exon 0N were identified by hierarchical cluster analysis of bisulfite sequencing data obtained from 710 alleles. Methylation at these centers was insignificant in normal epithelium, reached 80 to 90% in grade 4/5 PCa, but declined to less than 20% in bone metastases. In addition, progressive methylation spreading from the exonic CGI to the promoter CGI, which correlated with loss of ER-beta expression, was detected in microdissected samples and in cell cultures. Using a new class of methylated oligonucleotides that mediate sequence-specific methylation in cellulo, we demonstrated that methylation of the promoter CGI, but not the exonic CGIs, led to transcriptional inactivation of ER-beta. Our results present the first evidence that epigenetic regulation of ER-beta is a reversible and tumor stage-specific process and that gene silencing via methylated oligonucleotides may have therapeutic potential in the treatment of advanced PCa.

Topics: Base Sequence; Cell Line, Tumor; Dinucleoside Phosphates; DNA Methylation; DNA Primers; Estrogen Receptor beta; Humans; Male; Molecular Sequence Data; Neoplasm Metastasis; Neoplasm Staging; Precancerous Conditions; Promoter Regions, Genetic; Prostatic Neoplasms; Receptors, Estrogen; TATA Box

hDAB2IP (human DAB2 (also known as DOC-2) interactive protein) is a novel GTPase-activating protein for modulating the Ras-mediated signal pathway. We demonstrate that the down-regulation of hDAB2IP mRNA in prostate cancer (PCa) cells is regulated by transcriptional levels. Analysis of the hDAB2IP promoter revealed that it is a typical TATA-less promoter containing many GC-rich sequences. In this study, we delineated the potential impact of the epigenetic control of the hDAB2IP promoter on its gene regulation in PCa. Acetylhistone H3 was associated with the hDAB2IP promoter, and CpG islands remained almost unmethylated in normal prostatic epithelia, but not in PCa cell lines. Our data further indicated that trichostatin A (histone deacetylase inhibitor) and 5'-aza-2'-deoxycytidine (DNA hypomethylation agent) acted cooperatively in modulating hDAB2IP gene expression in PCa, whereas histone acetylation played a more significant role in this event. Moreover, a core promoter sequence from the hDAB2IP gene responsible for these treatments was identified. We therefore conclude that epigenetic regulation plays a potential role in regulating hDAB2IP expression in PCa and that these results also provide a new therapeutic strategy for PCa patients.

Topics: Base Sequence; Dinucleoside Phosphates; DNA Methylation; DNA Primers; DNA, Neoplasm; Epithelial Cells; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Humans; Male; Molecular Sequence Data; Promoter Regions, Genetic; Prostatic Neoplasms; ras GTPase-Activating Proteins; Reverse Transcriptase Polymerase Chain Reaction; TATA Box; Tumor Cells, Cultured

What defines the boundaries between methylated and unmethylated domains in the genome is unclear. In this study we used bisulfite genomic sequencing to map the boundaries of methylation that flank the 5'- and 3'-ends of the CpG island spanning the promoter region of the glutathione S-transferase (GSTP1) gene. We show that GSTP1 is expressed in a wide range of tissues including brain, lung, skeletal muscle, spleen, pancreas, bone marrow, prostate, heart, and blood and that this expression is associated with the CpG island being unmethylated. In these normal tissues a marked boundary was found to separate the methylated and unmethylated regions of the gene at the 5'-flank of the CpG island, and this boundary correlated with an (ATAAA)(19-24) repeated sequence. In contrast, the 3'-end of the CpG island was not marked by a sharp transition in methylation but by a gradual change in methylation density over about 500 base pairs. In normal tissue the sequences on either side of the 5'-boundary appear to lie in separate domains in which CpG methylation is independently controlled. These separate methylation domains are lost in all prostate cancer where GSTP1 expression is silenced and methylation extends throughout the island and spans across both the 5'- and 3'-boundary regions.

Topics: 5' Untranslated Regions; Brain; Dinucleoside Phosphates; DNA; DNA Methylation; Exons; Glutathione S-Transferase pi; Glutathione Transferase; Humans; Isoenzymes; Male; Molecular Sequence Data; Organ Specificity; Promoter Regions, Genetic; Prostate; Prostatic Neoplasms; Repetitive Sequences, Nucleic Acid; Transcription, Genetic

Advanced hormone-independent prostate cancer is characterized by a significant loss of androgen receptor (AR) expression in 20-30% of the tumors. The transcriptional block underlying this phenomenon is not known, but we have proposed that methylation of CpG sites in the AR promoter may reversibly inactivate transcription of the AR (D. F. Jarrard et al, Cancer Res., 58: 5310-5314, 1998). In this study, detailed methylation analysis using bisulfite sequencing was performed on a series of AR expression-positive and -negative prostate cancer cells. We found that methylation of several consensus sequences in the AR promoter (from -131 to -121 and +44 to +54) are tightly linked to the loss of AR expression in metastatic hormone-independent prostate cancer cell lines. These consensus sites of methylation correlate with the minimal promoter region critical for AR transcription. In human tissues, no methylation was demonstrated in normal or primary prostate cancers that express the AR. Four of 15 tumors obtained from men who had died from hormone-independent prostate cancer demonstrated a significant loss of AR expression immunohistochemically and two (50%) of these AR-negative tumors contained AR methylation. We conclude that the AR promoter contains specific CpG methylation hot spots that are markers for gene silencing. Furthermore, AR methylation may represent a phenotype important in the development of hormone independence in a subset of advanced prostate cancer in which AR expression is lost. The finding of AR methylation also represents the first report of aberrant methylation on an X-linked gene associated with a somatic male cancer.

Topics: 3' Untranslated Regions; Base Sequence; Consensus Sequence; Dinucleoside Phosphates; DNA Methylation; DNA Primers; DNA, Neoplasm; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Male; Promoter Regions, Genetic; Prostatic Neoplasms; Receptors, Androgen; Transcription, Genetic; Tumor Cells, Cultured

Epigenetic mechanisms including DNA methylation and histone deacetylation are thought to play important roles in gene transcriptional inactivation. Heterogenous expression of androgen receptor (AR), which appears to be related to variable responses to endocrine therapy in prostate cancer (PCa) may also be due to epigenetic factors. The methylation status of the 5' CpG island of the AR in 3 prostate cancer cell lines and 10 primary and 14 hormone-refractory PCa samples was determined using the bisulfite PCR methods. In DU145, CpG-rich regions of the AR were hypermethylated. By an immunohistochemical analysis, only one PCa sample had no AR expression, the others being heterogenous. Bisulfite sequencing and methylation-specific PCR analysis showed aberrant methylation of AR 5'-regulatory region in 20% of 10 primary and 28% of 14 hormone-refractory PCa samples. To clarify the effect of epigenetic regulation on AR expression, we treated three prostate cancer cell lines with a demethylating agent, 5-aza-2'-deoxycytidine (azaC), and a histone deacetylase inhibitor, Trichostatin A (TSA). In DU145, re-expression of AR mRNA was detected after treatment with azaC and/or TSA. Our results suggest that epigenetic regulations including CpG methylation and histone acetylation may play important roles in the regulation of the AR.

Topics: Antimetabolites, Antineoplastic; Azacitidine; Base Sequence; Binding Sites; Cyclic AMP Response Element-Binding Protein; Decitabine; Dinucleoside Phosphates; DNA Methylation; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Male; Polymerase Chain Reaction; Promoter Regions, Genetic; Prostatic Neoplasms; Receptors, Androgen; Tumor Cells, Cultured

Production of the potent vasoconstrictor endothelin-1 (ET-1) by human prostate cancer cells accompanies prostate cancer progression in vivo. The predominant endothelin receptor expressed by normal prostate epithelium, ETB, is not expressed by any of the established human prostate cancer cell lines, and ETB binding is decreased on prostate cancer tissues. ETB, which may mediate ET-1 clearance and may inhibit ET-1 secretion, is encoded by a gene that contains a 5' CpG island encompassing the transcriptional regulatory region. We examined this regulatory region of the ETB receptor gene (EDNRB) to determine whether hypermethylation of cytidine nucleotides accompanies decreased ETB expression in human prostate cancer. We found somatic methylation of CpG island sequences in EDNRB in 5 of 5 human prostate cancer cell lines, 15 of 21 primary prostate cancer tissues, and 8 of 14 prostate cancer metastases (70% of samples overall). Normal tissues contained only unmethylated EDNRB. Treatment of human prostatic carcinoma cell line cultures with 5-azacytidine induced ETB mRNA expression, suggesting that CpG island methylation changes might accompany the apparent transcriptional silencing of EDNRB in vivo.

Topics: Dinucleoside Phosphates; Humans; Male; Methylation; Prostatic Neoplasms; Receptor, Endothelin B; Receptors, Endothelin; Regulatory Sequences, Nucleic Acid; Tumor Cells, Cultured

P-Cadherin is a member of the cadherin family of cell surface glycoproteins that mediate Ca2+-dependent cell-cell adhesion and is expressed in a differential fashion in normal epithelial tissues. The expression of P-cadherin in human prostate cancer development has not been investigated previously. By immunohistochemistry, we show that P-cadherin expression is restricted to the cell-cell border of basal epithelial cells in 30 normal prostate samples. This staining is down-regulated in prostatic intraepithelial neoplasia and is absent in all 25 of the well to poorly differentiated prostate cancer specimens analyzed. To examine potential P-cadherin-regulatory elements, we sequenced the 5'-flanking region of this gene. Similar to the mouse gene, the human P-cadherin promoter is TATA-less, contains an Sp-1 binding site and, analogous to the human E-cadherin sequence, demonstrates a GC-rich region characteristic of a CpG island. Cytosine methylation of this region occurs in P-cadherin-negative prostate cancer cell lines but not in cell lines expressing this gene. In vivo, a lack of expression in 12 clinical prostate cancer specimens is not associated with methylation of the P-cadherin promoter. These results demonstrate that the expression of the basal cell marker P-cadherin is lost in prostate cancer development and that in vivo mechanisms other than cytosine methylation regulate this consistent loss of expression.

Topics: Animals; Base Composition; Binding Sites; Biomarkers; Cadherins; Dinucleoside Phosphates; Epithelial Cells; Humans; Immunohistochemistry; Male; Mice; Promoter Regions, Genetic; Prostate; Prostatic Neoplasms; Restriction Mapping; Reverse Transcriptase Polymerase Chain Reaction; Sp1 Transcription Factor; Tumor Cells, Cultured

The recently identified cyclin-dependent kinase inhibitor p15INK4B is localized to a region on chromosome 9p21 frequently deleted in human tumors. Previous evidence has pointed to a related gene, p16INK4A, as the principal target of this deletion. We report that in gliomas and, to a striking degree, in leukemias, the p15 gene is commonly inactivated in association with promoter region hypermethylation involving multiple sites in a 5'-CpG island. In some gliomas and all of the primary leukemias, this event occurs without alteration of the adjacent gene, p16INK4A. In other tumors, including lung, head and neck, breast, prostate, and colon cancer, inactivation of p15INK4B occurs only rarely and only with concomitant inactivation of p16. Aberrant methylation of p15INK4B is associated with transcriptional loss of this gene. Treatment with the demethylating agent 5-aza-2'-deoxycytidine leads to re-expression of p15 mRNA. In selected leukemia cell lines, p15 inactivation correlates with known resistance to the growth-suppressive effects of transforming growth factor-beta. These results suggest that p15INK4B is inactivated selectively in leukemias and gliomas and seems to constitute an important tumor suppressor gene loss in these neoplasms.

Topics: Adult; Breast Neoplasms; Carrier Proteins; Cell Cycle Proteins; Cell Line; Child; Chromosome Deletion; Chromosome Mapping; Chromosomes, Human, Pair 9; Colonic Neoplasms; Cyclin-Dependent Kinase Inhibitor p15; Cyclin-Dependent Kinase Inhibitor p16; Dinucleoside Phosphates; DNA; DNA, Neoplasm; Female; Genes, Tumor Suppressor; Glioma; Homozygote; Humans; Leukemia; Leukemia, Myeloid, Acute; Lung Neoplasms; Lymphocytes; Male; Methylation; Polymerase Chain Reaction; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Prostatic Neoplasms; Reference Values; Restriction Mapping; Tumor Cells, Cultured; Tumor Suppressor Proteins