Lentiviral vector neutral endopeptidase gene transfer suppresses prostate cancer tumor growth

Neprilysin (neutral endopeptidase, NEP) is a cell surface peptidase whose expression is lost in approximately 50% of prostate cancers (PC). NEP normally functions to inactivate peptides such as bombesin and endothelin-1, and potentiates the effects of the PTEN tumor suppressor via a direct protein-protein interaction. NEP loss contributes to PC progression. We investigated the therapeutic efficacy of using a lentiviral vector system to restore NEP expression in PC cells. Third-generation lentiviral vectors encoding wild-type NEP (L-NEP) or green fluorescent protein (L-GFP) were introduced into NEP-deficient 22RV1 PC cells. Cells infected with L-NEP or L-GFP at a multiplicity of infection of 10 demonstrated NEP enzyme activity of 1171.2+/-4.9 and 17.2+/-5.3 pmol/microg/min (P<0.0001), respectively. Cell viability, proliferation and invasion were each significantly inhibited in 22RV1 cells expressing NEP compared with control cells infected with L-GFP (P<0.01). Analysis of known downstream effects of NEP showed NEP-expressing cells exhibiting decreased Akt and focal adhesion kinase phosphorylation and increased PTEN protein expression. Finally, injection of L-NEP into established 22RV1 xenograft tumors significantly inhibited tumor growth (P<0.01). These experiments demonstrate that lentiviral NEP gene transfer is a novel targeted strategy for the treatment of NEP-deficient PC.     

Tumor-suppressive effects of neutral endopeptidase in androgen-independent prostate cancer cells.

Expression of neutral endopeptidase (NEP) 24.11 is diminished in metastatic, androgen-independent prostate cancers (PCs; C. N. Papandreou et al., NAT: MED:, 4: 50--57, 1998). To determine the effects on androgen-independent PC cells of overexpressing cell-surface NEP, an inducible tetracycline-regulatory gene expression system was used to stably introduce and express the NEP gene in androgen-independent TSU-Pr1 cells generating WT-5 cells, which expressed high levels of enzymatically active NEP protein when cultured in the absence of tetracycline. TN12 cells, which contain the identical vectors without the NEP gene and do not express NEP, were used as control. Expression of NEP in WT-5 cells after removal of tetracycline from the media resulted in a >80% inhibition in cell proliferation over a 1-week period (P < 0.005) compared with control cells. Tumor formation occurred in the prostate glands of orthotopically injected athymic mice killed at 30 days in 4 of 5 mice that were given injections of 2 x 10(6) WT-5 cells and were fed doxycycline (NEP suppressed), and in all mice that were given injections of TN12 cells and were fed with or without doxycycline. In contrast, only 1 of 5 mouse prostates developed a tumor in mice that were given injections of WT-5 cells and that did not receive doxycycline. Analysis of the mechanisms of NEP-induced growth suppression revealed that NEP expression in WT-5 cells induced a 4-fold increase in the number of PC cells undergoing apoptosis, and increased the expression of p21 tumor suppressor gene protein and the level of unphosphorylated retinoblastoma protein as determined by Western blot. Flow cytometric analysis show that induced NEP expression in WT-5 cells resulted in a G(1) cell cycle arrest. These data show that NEP can inhibit PC cell growth and tumorigenicity and suggest that NEP has potential as therapy for androgen-independent PC.     

Inactivation of cyclin D2 gene in prostate cancers by aberrant promoter methylation.

PURPOSE: Loss or abnormal expression of Cyclin D2, a crucial cell cycle-regulatory gene, has been described in human cancers; however, data for prostate tumors are lacking. We investigated the epigenetic silencing of Cyclin D2 gene in prostate cancers and correlated the data with clinicopathological features. EXPERIMENTAL DESIGN: Cyclin D2 promoter methylation was analyzed in 101 prostate cancer samples by methylation-specific PCR. In addition, we analyzed 32 nonmalignant prostate tissue samples, which included 24 samples of benign disease, benign prostatic hypertrophy, or prostatitis and 7 normal tissues adjacent to cancer. The methylation status of Cyclin D2 was correlated with the methylation of nine other tumor suppressor genes published previously from our laboratory on the same set of samples (R. Maruyama et al., Clin. Cancer Res., 8: 514-519, 2002). The methylation index was determined as a reflection of the methylated fraction of the genes examined. RESULTS: The frequency of methylation of Cyclin D2 promoter was significantly higher in prostate cancers (32%) than in nonmalignant prostate tissues (6%; P = 0.004), and it was not age related. Aberrant methylation was present at insignificant levels in peripheral blood lymphocytes (8%). We also compared methylation of cyclin D2 with methylation of nine tumor suppressor genes [published previously from our laboratory (R. Maruyama et al., Clin. Cancer Res., 8: 514-519, 2002)] studied in the same set of samples. The concordances between methylation of Cyclin D2 and the methylation of RARbeta, GSTP1, CDH13, RASSF1A, and APC were statistically significant, whereas methylation of P16, DAPK, FHIT, and CDH1 were not significant. The differences in methylation index between malignant and nonmalignant tissues for all 10 genes were statistically significant (P < 0.0001). Among clinicopathological correlations, the high Gleason score group had significantly greater methylation frequency of Cyclin D2 (42%; P = 0.004). Although the high preoperative serum prostate-specific antigen (PSA) group did not have significantly greater methylation frequency, methylation of Cyclin D2 had higher mean PSA value. Also, the prostate cancers in the high Gleason score group had high mean values of PSA. CONCLUSIONS: Our results indicate that methylation of Cyclin D2 in prostate cancers correlates with clinicopathological features of poor prognosis. These findings are of biological and potential clinical importance.     

基因启动子甲基化和化疗耐药

表观遗传（eprgenetic）和基因突变、易位、缺失等事件同样在化疗耐药的形成中有重要的作用。表观遗传的重要机制之一-基因的甲基化是细胞中必不可少的一种修饰方式，正常人体细胞大约有29000个CpG岛，并以非随机化的方式主要分布于蛋白编码基因的启动子和第一外显子区域。但是肿瘤细胞中基因甲基化状态有所改变，表现为整体甲基化程度降低和局部甲基化程度的增高，而这些改变进而可以影响肿瘤细胞对于化疗药物的敏感性。我们就基因启动子的异常甲基化和化疗耐药之间的关系作一探讨，并对近来一些基因甲基化及其逆转的研究进行简要回顾。     

鼻咽癌组织中DAPK基因启动子甲基化的研究

目的:分析鼻咽癌组织中DAPK基因启动子的甲基化状态,探讨DAPK基因启动子甲基化与鼻咽癌的关系。方法:应用甲基化特异性PCR技术检测48例鼻咽癌组织、26例慢性鼻咽黏膜炎症组织的DAPK基因启动子甲基化状态,比较鼻咽癌和慢性鼻咽黏膜炎症组织的DAPK基因启动子甲基化率。结果:鼻咽癌组织的DAPK基因启动子甲基化率为75%,而慢性鼻咽黏膜炎症组织中未检测到DAPK基因启动子甲基化。结论:鼻咽癌组织中DAPK基因启动子存在高甲基化水平,检测DAPK基因启动子甲基化或许能为鼻咽癌的诊断提供依据。     

Methylation and silencing of the Thrombospondin-1 promoter in human cancer.

Neovascularization is a common feature of many human cancers, but relatively few molecular defects have been demonstrated in genes regulating angiogenesis. Decreased expression of Thrombospondin-1 (THBS1), a P53 and Rb regulated angiogenesis inhibitor, has been observed in some human tumors, including glioblastoma multiforme (GBM). To study whether methylation-associated inactivation is involved in down-regulating THBS1 expression in cancer, we analysed the methylation status of THBS1 in several cell lines and primary tumors. Three cell lines (RKO, CEM and RAJI) were completely methylated at several CpG sites within the THBS1 5' CpG island, and had no detectable expression by RT-PCR. THBS1 expression was readily reactivated using the methylation-inhibitor 5-deoxy-azacytidine in all three lines. Furthermore, THBS1 methylation was present in 33% (14/42) of primary GBMs. Thus, de novo methylation may serve as a potential way to inactivate THBS1 expression in human neoplasms.     

Methylation status of oestrogen receptor-alpha gene promoter sequences in human ovarian epithelial cell lines

We have determined the methylation status of the CpG island of the oestrogen receptor alpha gene in seven human ovarian cell lines. Cell lines expressing oestrogen receptor alpha showed no evidence of hypermethylation. In three of four cell lines that produced no detectable oestrogen receptor alpha protein, hypermethylation was observed at the NotI site of the CpG island. These results indicate that aberrant hypermethylation may be responsible for a significant proportion of epithelial ovarian tumours in which oestrogen receptor alpha expression is lost.     

Methylation pattern of CDH13 gene in digestive tract cancers

Recently, the loss of CDH13 (T-cadherin, H-cadherin) gene expression accompanied by CDH13 promoter methylation was identified in colon cancers. We examined CDH13 methylation in oesophageal and gastric carcinomas. Five of 37 oesophageal cancers (14%) and 23 of 66 gastric cancers (35%) demonstrated abnormal methylation of the CDH13 promoter. Abnormal methylation was frequently found in gastric cancers of patients at all clinical stages just as in E-cadherin, another of the cadherin family, suggesting that these cancers could be methylated at an early stage. These results suggested that CDH13 might play a variety of roles depending on the tissue type.     

Inactivation of the putative suppressor gene DOK1 by promoter hypermethylation in primary human cancers

The DOK1 gene is a putative tumour suppressor gene located on the human chromosome 2p13 which is frequently rearranged in leukaemia and other human tumours. We previously reported that the DOK1 gene can be mutated and its expression down-regulated in human malignancies. However, the mechanism underlying DOK1 silencing remains largely unknown. We show here that unscheduled silencing of DOK1 expression through aberrant hypermethylation is a frequent event in a variety of human malignancies. DOK1 was found to be silenced in nine head and neck cancer (HNC) cell lines studied and DOK1 CpG hypermethylation correlated with loss of gene expression in these cells. DOK1 expression could be restored via demethylating treatment using 5-aza-2'deoxycytidine. In addition, transduction of cancer cell lines with DOK1 impaired their proliferation, consistent with the critical role of epigenetic silencing of DOK1 in the development and maintenance of malignant cells. We further observed that DOK1 hypermethylation occurs frequently in a variety of primary human neoplasm including solid tumours (93% in HNC, 81% in lung cancer) and haematopoietic malignancy (64% in Burkitt's lymphoma). Control blood samples and exfoliated mouth epithelial cells from healthy individuals showed a low level of DOK1 methylation, suggesting that DOK1 hypermethylation is a tumour specific event. Finally, an inverse correlation was observed between the level of DOK1 gene methylation and its expression in tumour and adjacent non tumour tissues. Thus, hypermethylation of DOK1 is a potentially critical event in human carcinogenesis, and may be a potential cancer biomarker and an attractive target for epigenetic-based therapy.     

Aberrant promoter methylation of insulin-like growth factor binding protein-3 gene in human cancers

Insulin-like growth factor binding protein-3 (IGFBP-3) is postulated to be a mediator of growth suppression signals. Here, we examined the methylation status of IGFBP-3 to correlate to clinicopathological factors in human cancers. The methylation status of IGFBP-3 was determined by bisulfite DNA sequencing and was correlated with expression semi-quantified by real-time RT-PCR to develop a methylation-specific PCR (MSP) assay for IGFBP-3. Using the MSP assay, we examined the methylation status of IGFBP-3 in gastric cancer (GC), colorectal cancer (CRC), breast cancer (BC) and malignant mesothelioma (MM). IGFBP-3 methylation was detected in 6 of 13 (46%) and 16 of 24 (67%) GC cell lines and tumors, respectively; 4 of 8 (50%) and 15 of 26 (58%) CRC cell lines and tumors, respectively; 3 of 11 (27%) and 7 of 39 (18%) BC cell lines and tumors, respectively and 1 of 5 (20%) and 18 of 56 (32%) MM cell lines and tumors, respectively. Interestingly, the methylation status of MM specimens from Japanese patients (75%, 12 out of 16 patients) was significantly higher than those from the USA (15%, 6 out of 40 patients) (p < 0.0001), suggesting the presence of ethnic differences in the IGFBP-3 methylation status. We also found that IGFBP-3 methylation was preferentially present in GCs arising in the lower-third of the stomach (p = 0.079). In summary, our results showed that IGFBP-3 methylation played an important role in the silencing of its expression, suggesting that IGFBP-3 may act as a tumor suppressor gene in several human cancers examined.     

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.     

Methylation status of insulin-like growth factor-binding protein-3 promoter in prostate cancer tissues

Background: It has been postulated that insulin‐like growth factor‐binding protein‐3 (IGFBP‐3) is a mediator of growth suppression signals. Recently, a correlation between the promoter hypermethylation of IGFBP‐3 and cancer risk was reported in hepatocellular cancer and in non‐small‐cell lung cancer (NSCLC). We investigated whether the methylation status of IGFBP‐3 promoter in prostate tissues influences the progression and prognosis of prostate cancer. Methods: We conducted a case‐control study consisting of 38 prostate cancer patients and 57 control subjects, and assessed the relationship of promoter hypermethylation at IGFBP‐3 with the clinical and pathological tumor characteristics of these patients. The methylation status was analyzed by two methylation‐specific PCR techniques. Results: Hypermethylation of the IGFBP‐3 promoter was found in 13 (34.2%) of the 38 cases, and 11 (19.3%) of the 57 controls with one method using a hepatocellular primer as described by Hanafusa et al. With other methods using a NSCLC‐primer, as described by Chang et al., hypermethylation was found in four (10.5%) of the cases and two (3.5%) of the controls. No significant differences were observed in the methylation frequencies between the controls and the cases. When the cases were stratified based on their clinical stage (localized or metastatic) and pathological grade (Gleason score of <7 or ≥7), the promoter hypermethylation status of IGFBP‐3 also showed no significant differences. Conclusions: Hypermethylation of the IGFBP‐3 promoter was observed in prostate tissues, but we found no significant association of IGFBP‐3 promoter hypermethylation with the risk of developing prostate cancer.