Protein phosphatase and TRAIL receptor genes as new candidate tumor genes on chromosome 8p in prostate cancer.

BACKGROUND: Allelic losses on chromosome 8p are common in prostate carcinoma, but it is not known exactly how they contribute to cancer development and progression. MATERIALS AND METHODS: Expression of 12 genes located across chromosome 8p, including established tumor suppressor candidates (CSMD1, DLC1, NKX3.1), and others from a new microarray-based comparison was studied by quantitative RT-PCR in 45 M0 prostate carcinomas and 13 benign prostate tissues. RESULTS: Significantly reduced expression was observed for two protein phosphatase subunit genes (PPP2CB, PPP3CC) and two TRAIL decoy receptors (TNFRSF10C/DcR1, TNFRSF10D/DcR2), but not for the three established candidates nor for TRAIL death receptor genes. Low expression of PPP3CC and TNFRSF10C located at 8p21.3 was highly significantly associated with tumor recurrence. In addition to allele loss, down-regulation of TNFRSF10C and TNFRSF10D was found to be associated with hypermethylation, although bisulfite sequencing usually revealed it to be partial. CONCLUSION: Our data strongly support a recent proposal that a segment at 8p21.3 contains crucial prostate cancer tumor suppressors. In addition, they raise the paradoxical issue of why TRAIL decoy receptors rather than death receptors are down-regulated in aggressive prostate cancer.

Clinical and functional results after continent cutaneous urinary diversion with the ileal double-T-pouch.

INTRODUCTION: The aim of the study was to assess the clinical and functional results after continent cutaneous diversion with the ileal double-T-pouch. PATIENTS AND METHODS: Between July 1998 and July 2006, 19 patients underwent continent urinary diversion with a cutaneous ileal double-T-pouch. Follow-up investigations included blood chemistry, renal ultrasound and evaluation of the outlet function. The median follow-up was 24 months (range 3-76). RESULTS: There were no intraoperative complications and no perioperative mortality. Early postoperative complications not related to urinary diversion occurred in 6 patients (31.6%). Five patients developed complications related to the urinary diversion (26.3%). The mean operating time was 580 min (420-840). Including the patients who had good results after reoperation, 16 out of the 19 patients (84.2%) were continent day and night without any catheterization difficulties. The average pouch capacity was 490 ml (range 250-1,100). A mild acidosis was the only metabolic disorder observed. During follow-up renal function remained stable in all patients, urinary reflux, recurrent pyelonephritis or stone formation were not found. Three patients with a body mass index >30 developed a necrosis of the efferent loop resulting in pouch cutaneous fistulas. CONCLUSIONS: Continent cutaneous urinary diversion with the ileal double-T-pouch led to good intermediate functional and clinical results. However, construction of the pouch is sophisticated, resulting in a long operation time. The double-T-pouch is not recommended in obese patients.

Downregulation of several fibulin genes in prostate cancer.

BACKGROUND: Fibulins, encoded by FBLN genes, are extracellular matrix proteins influencing cell adhesion and migration. Altered expression of fibulins is associated with progression of several cancer types, but has not been studied in prostate cancer. METHODS: Expression of FBLN1 (major splice forms C and D), FBLN4, FBLN5, SPOCK1, and TENC was compared between 47 prostate cancer samples and 13 benign prostatic tissues by quantitative RT-PCR. Fibulin-1 and fibulin-5 expression was studied by immunohistochemistry. Effects of androgens and the DNA methylation inhibitor 5-aza-2'-deoxycytidine on fibulin expression were investigated in different prostate cancer cell lines. RESULTS: Our recent microarray analysis suggested downregulation of three fibulins, FBLN1, FBLN4, and FBLN5, in prostate cancer, while two further ECM genes, SPOCK1 (testican) and TENC (tenascin C), appeared upregulated or unchanged. These observations were corroborated by quantitative RT-PCR. Accordingly, FBLN1 and FBLN4 were weakly expressed in carcinoma lines compared to normal prostate epithelial cells (PrECs). Only FBLN4 was induced by 5-aza-2'-deoxycytidine, but its promoter was unmethylated. Androgen did not affect expression of FBLN genes. The FBLN1C and FBLN1D splice forms were coordinately expressed. Fibulin-1 protein was weakly detectable in benign PrECs, but tended to accumulate in cancer cells. Fibulin-5 was predominantly located in the stroma with a strong gradient from the periurethral to the peripheral zone, and lost in cancers. CONCLUSIONS: Three FBLN genes are significantly downregulated in prostate cancer, whereas SPOCK1 is often upregulated. FBLN5 downregulation fits its postulated anticancerous function, whereas FBLN1 and FBLN4 behave different than in certain other cancers.

A new and reliable culture system for superficial low-grade urothelial carcinoma of the bladder.

Several bladder cancer culture systems have been developed in recent years. However, reports about successful primary cultures of superficial urothelial carcinomas (UC) are sparse. Based on the specific growth requirements of UC described previously, we developed a new and reliable culture system for superficial low-grade UC. Between November 2002 and April 2006, 64 primary cultures of bladder cancer specimens were performed. After incubating the specimens overnight in 0.1% ethylenediaminetetraacetic acid solution, tumour cells could easily be separated from the submucosal tissue. Subsequently, cells were seeded in a low-calcium culture medium supplemented with 1% serum, growth factors, non-essential amino acids and glycine. The malignant origin of the cultured cells was demonstrated by spectral karyotyping. Overall culture success rate leading to a homogenous tumour cell population without fibroblast contamination was 63%. Culture success could be remarkably enhanced by the addition of glycine to the culture medium. Interestingly, 86.4% of pTa tumours were cultured successfully compared to only 50% of the pT1 and 38% of advanced stage tumours, respectively. G1 and G2 tumours grew significantly better than G3 tumours (86, 73 and 41%, respectively). Up to three passages of low-grade UC primary cultures were possible. We describe a new and reliable culture system, which is highly successful for primary culture and passage of low-grade UC of the bladder. Therefore, this culture system can widely be used for functional experiments on early stage bladder cancer.

Factor interaction analysis for chromosome 8 and DNA methylation alterations highlights innate immune response suppression and cytoskeletal changes in prostate cancer.

BACKGROUND: Alterations of chromosome 8 and hypomethylation of LINE-1 retrotransposons are common alterations in advanced prostate carcinoma. In a former study including many metastatic cases, they strongly correlated with each other. To elucidate a possible interaction between the two alterations, we investigated their relationship in less advanced prostate cancers. RESULTS: In 50 primary tumor tissues, no correlation was observed between chromosome 8 alterations determined by comparative genomic hybridization and LINE-1 hypomethylation measured by Southern blot hybridization. The discrepancy towards the former study, which had been dominated by advanced stage cases, suggests that both alterations converge and interact during prostate cancer progression. Therefore, interaction analysis was performed on microarray-based expression profiles of cancers harboring both alterations, only one, or none. Application of a novel bioinformatic method identified Gene Ontology (GO) groups related to innate immunity, cytoskeletal organization and cell adhesion as common targets of both alterations. Many genes targeted by their interaction were involved in type I and II interferon signaling and several were functionally related to hereditary prostate cancer genes. In addition, the interaction appeared to influence a switch in the expression pattern of EPB41L genes encoding 4.1 cytoskeleton proteins. Real-time RT-PCR revealed GADD45A, MX1, EPB41L3/DAL1, and FBLN1 as generally downregulated in prostate cancer, whereas HOXB13 and EPB41L4B were upregulated. TLR3 was downregulated in a subset of the cases and associated with recurrence. Downregulation of EPB41L3, but not of GADD45A, was associated with promoter hypermethylation, which was detected in 79% of carcinoma samples. CONCLUSION: Alterations of chromosome 8 and DNA hypomethylation in prostate cancer probably do not cause each other, but converge during progression. The present analysis implicates their interaction in innate immune response suppression and cytoskeletal changes during prostate cancer progression. The study thus highlights novel mechanisms in prostate cancer progression and identifies novel candidate genes for diagnostic and therapeutic purposes. In particular, TLR3 expression might be useful for prostate cancer prognosis and EPB41L3 hypermethylation for its detection.

Expression changes in EZH2, but not in BMI-1, SIRT1, DNMT1 or DNMT3B are associated with DNA methylation changes in prostate cancer.

The polycomb proteins BMI-1, EZH2, and SIRT1 are characteristic components of the PRC1, PRC2, and PRC4 repressor complexes, respectively, that modify chromatin. Moreover, EZH2 may influence DNA methylation by direct interaction with DNA methyltransferases. EZH2 expression increases during prostate cancer progression, whereas BMI-1 and SIRT1 are not well investigated. Like EZH2 expression, DNA methylation alterations escalate in higher stage prostate cancers, raising the question whether these epigenetic changes are related. Expression of EZH2, BMI-1, SIRT1, and the DNA methyltransferases DNMT1 and DNMT3B measured by qRT-PCR in 47 primary prostate cancers was compared to APC, ASC, GSTP1, RARB2, and RASSF1A hypermethylation and LINE-1 hypomethylation. SIRT1 and DNMT3B were overexpressed in cancerous over benign tissues, whereas BMI-1 was rather downregulated and DNMT1 significantly diminished. Nevertheless, cancers with higher DNMT1 and BMI-1 expression had worse clinical characteristics, as did those with elevated EZH2. In particular, above median DNMT1 expression predicted a worse prognosis. EZH2 and SIRT1 overexpression were well correlated with increased MKI67. Immunohistochemistry confirmed limited EZH2 and heterogeneous DNMT3B overexpression and explained the decrease in BMI-1 by pronounced heterogeneity among tumor cells. EZH2 overexpression, specifically among all factors investigated, was associated with more frequent hypermethylation, in particular of GSTP1 and RARB2, and also with LINE-1 hypomethylation. Our data reveal complex changes in the composition of polycomb repressor complexes in prostate cancer. Heterogeneously expressed BMI-1 and slightly increased EZH2 may characterize less malignant cancers, whereas more aggressive cases express both at higher levels. SIRT1 appears to be generally increased in prostate cancers. Intriguingly, our data suggest a direct influence of increased EZH2 on altered DNA methylation patterns in prostate cancer.

Epigenetic control of CTCFL/BORIS and OCT4 expression in urogenital malignancies.

Aberrant hypomethylation in many cancers reactivates retrotransposons and selected single-copy genes such as cancer-testis antigens. Genes reactivated in this manner have recently been postulated to include CTCFL/BORIS, a presumptive testis-specific chromatin regulator, and OCT4/POU5F1, a transcriptional activator in pluripotent cells. We found both genes expressed at high levels in testis and at much lower levels in normal prostate tissue. In prostate and bladder carcinoma cell lines and cancer tissues expression remained largely unchanged, but individual prostate carcinomas showed modestly increased CTCFL expression compared to normal tissues. OCT4 expression was significantly decreased in cancer tissues. Promoter methylation in both genes paralleled expression levels. CTCFL, but not OCT4 was dramatically induced in cancer cell lines by 5-aza-2'-deoxycytidine, but neither gene by the histone deacetylase inhibitor suberoylanilide hydroxamic acid. Thus, CTCFL and OCT4 resemble cancer-testis antigens in being selectively hypomethylated and expressed in male germ cells but differ in lacking significant reexpression and hypomethylation in prostate carcinomas. DNA methylation appears the crucial mechanism in the control of CTCFL transcription, but less decisive in that of OCT4. These findings imply that inhibitors of DNA methylation used for cancer treatment may induce CTCFL expression. Immunohistochemistry demonstrated nuclear localization of CTCFL in developing spermatocytes, and cytoplasmatic localization in spermatogonia, Leydig cells, and epithelial prostate cells. Teratocarcinoma cell lines showed nuclear, and 5-aza-2'-deoxycytidine-treated prostate cancer lines nuclear or cytoplasmatic localization. These different localizations might indicate additional control of CTCFL function via intracellular compartmentation.

Genomic and expression analysis of the 3q25-q26 amplification unit reveals TLOC1/SEC62 as a probable target gene in prostate cancer.

Gain at chromosome 3q25-q26 has been reported to commonly occur in prostate cancer. To map the 3q25-q26 amplification unit and to identify the candidate genes of amplification, we did fluorescence in situ hybridization and quantitative real-time PCR for gene copy number and mRNA expression measurements in prostate cancer cell lines and prostate cancer samples from radical prostatectomy specimens. The minimal overlapping region of DNA copy number gains in the cell lines could be narrowed down to 700 kb at 3q26.2. Of all positional and functional candidates in this region, the gene TLOC1/SEC62 revealed the highest frequency (50%) of copy number gains in the prostate cancer samples and was found to be up-regulated at the mRNA level in all samples analyzed. TLOC1/Sec62 protein was also shown to be overexpressed by Western blot analysis. Intriguingly, the TLOC1/SEC62 gene copy number was increased in prostate tumors from patients who had a lower risk of and a longer time to progression following radical prostatectomy. These findings make TLOC1/SEC62 the best candidate within the 3q amplification unit in prostate cancer. TLOC1/Sec62 protein is a component of the endoplasmic reticulum protein translocation machinery, whose function during prostate carcinogenesis remains to be determined.

Relationship of NKX3.1 and MYC gene copy number ratio and DNA hypomethylation to prostate carcinoma stage.

OBJECTIVE: High stage prostate cancers have been reported to frequently harbor chromosome 8 alterations and hypomethylation of LINE-1 retrotransposons. The potential of these parameters for molecular staging of prostate carcinoma was investigated. METHODS: High molecular weight DNA was extracted from 63 carcinoma tissues (22 pT2, 38 pT3, 3 pT4). Chromosome 8 alterations were followed by determining the ratio of NKX3.1 (at 8p21) to MYC (at 8q24) gene copy numbers (NKX3.1:MYC ratio) using a new real-time PCR technique. LINE-1 hypomethylation was quantified by Southern blot analysis. RESULTS: In 42 carcinomas NKX3.1 copy numbers were altered, with decreases in 32 cases. Copy numbers of MYC were increased in 38 cases and diminished in four. The NKX3.1:MYC ratio was altered in 45 specimens, with a decrease in all but two. NKX3.1 loss was associated with tumor stage (p<0.03) and MYC gain with Gleason score (p<0.03). The NKX3.1:MYC ratio was highly significantly associated with tumor stage (p<0.002), displaying 66% sensitivity and 87% specificity. LINE-1 hypomethylation was related (p<0.004) to tumor stage, but exhibited lower sensitivity (59%) and specificity (77%). CONCLUSION: A straightforward PCR technique detecting chromosome 8 alterations might be useful to predict which prostate cancers are organ-confined while determination of hypomethylation appears to be somewhat less well suited.

Hypomethylation of the XIST gene promoter in prostate cancer.

In a process denoted "global hypomethylation" repetitive DNA sequences like LINE-1 retrotransposons become hypomethylated in human cancers, including a subset of prostate carcinomas. It is less well known to what extent single-copy sequences are affected by this phenomenon. Therefore, we have analyzed methylation and expression of the XIST gene by bisulfite sequencing and real-time RT-PCR. The promoter of this single-copy gene is strongly methylated in normal male cells, including leukocytes and normal prostate. In prostate cancer tissues and particularly in cell lines, partial hypomethylation was observed paralleling that of LINE-1 sequences. Weak XIST expression was found in normal prostate tissues, but none in leukocytes. Only slight increases in expression of this gene were found in cancer tissues and cell lines. Our data suggest that hypomethylation in prostate cancer is indeed "global," affecting repeat and unique sequences in parallel. Detection of partially hypomethylated XIST alleles in prostate cancer tissues might be useful for the identification of cases with pronounced hypomethylation, which tend to be more aggressive.

Clinical-scale generation of dendritic cells in a closed system.

Immunotherapy of malignant diseases based on dendritic cells (DCs) pulsed with tumor antigens is a promising approach. Therefore, there is a demand for large-scale, clinical-grade ex vivo generation of DCs. Here, a procedure is presented that combines monocyte selection and tissue culture in closed systems under current good manufacturing practice conditions. Leukocytes from three patients with urologic cancers were collected by leukapheresis and subjected to immunomagnetic enrichment. From leukapheresis products containing 1.6 +/- 0.2 x 1010 (mean +/- SEM) leukocytes with a frequency of CD14+ monocytes of 18.7 +/- 2.3%, monocytes were enriched to 94.3 +/- 2.2%. CD14+ cell recovery was 67.0 +/- 4.7%. After 6 days of culture in Teflon bags in X-Vivo 15 medium supplemented with autologous plasma, GM-CSF, and IL-4, cells showed an immature DC phenotype and efficient antigen uptake. Following an additional 3 days of culture in the presence of GM-CSF, IL-4, IL-1beta, IL-6, TNFalpha, and PGE(2), cells (82.0 +/- 5.8% CD83+) displayed a mature DC morphology and phenotype, including expression of CD11b, CD11c, CD18, CD25, CD40, CD54, CD58, CD80, CD86, HLA class I, and HLA-DR as well as expression of CCR7 but not CCR5. The mature DC phenotype remained stable for at least 5 days in the absence of cytokines. Yield of DC was 14.0 +/- 4.7% and viability was 91.9 +/- 3.5%. Mature DCs effectively clustered with naive T cells and potently induced allogeneic T-cell proliferation and IL-2 and IFNgamma but not IL-4 production. Thus, this procedure allows large-scale generation of stably mature, Th1 responses inducing DCs under cGMP conditions in a closed system from cancer patients and is therefore well suited for immunotherapy.