Candidate tumor suppressor gene SLC5A8 is frequently down-regulated by promoter hypermethylation in prostate tumor

Background

The prostate gland is the most common site of cancer and the third leading cause of cancer mortality in men. Solute carrier family 5 (iodide transporter), member 8 (SLC5A8) was proposed as a potential tumor suppressor gene which is silenced by epigenetic changes in various tumors. The aim of this study was to investigate the significance of DNA methylation in SLC5A8 expression in prostate tumors.

Methods

DNA methylation status of the promoter region and expression of SLC5A8 were evaluated in prostate cancer cell lines, tumor and adjacent non-tumor prostate tissues from same prostate cancer patients, by using bisulphite-modified sequencing, RT-PCR and quantitative methylation-specific PCR (QMSP) analysis.

Results

The reduced or lost expression of SLC5A8 was observed in 70% of the tumor tissues. The bisulphite-modified sequencing analysis on the prostate cancer cell lines which do not express SLC5A8 detected the densely methylated SLC5A8 promoter region. SLC5A8 was reactivated by treatment with DNA methyl transferase inhibitor, 5-azacytidine but not by trichostatin A (TSA). Higher methylation at the promoter region of SLC5A8 in primary prostate tumor tissues was detected as compared with those in adjacent non-tumor tissues (7/10, 70%).

Conclusions

These data suggested that DNA methylation in the SLC5A8 promoter region suppressed the expression of SLC5A8 in prostate tumor.     

Methylation of a CpG island within the promoter region of the KAI1 metastasis suppressor gene is not responsible for down-regulation of KAI1 expression in invasive cancers or cancer cell lines

The molecular basis for downregulation of the KAI1 metastasis suppressor gene in invasive and metastatic human cancers is unknown. We have used bisulphite methylation analysis of DNA from paraffin-embedded invasive bladder tumour samples and from bladder cancer cell lines to determine if hypermethylation of a CpG island within the KAI1 promoter is responsible for this effect. Representative invasive tumour cell lines were also exposed to 5-aza-2-deoxycytidine. We found no evidence for hypermethylation of the CpG island and suggest that mechanisms other than promoter hypermethylation are responsible for reduced KAI1 expression in invasive bladder tumours and tumour cell lines.     

CpG methylation at promoter site -140 inactivates TGFbeta2 receptor gene in prostate cancer

BACKGROUND: The action of transforming growth factor beta (TGF-beta) is mediated through type 1 (TbetaRI) and type 2 (TbetaRII) receptors. Prostate cancer cells are often resistant to TGF-beta signaling due to loss of TbetaRII expression. The authors of the current study hypothesized that CpG methylation of the TbetaRII promoter at the Sp1 binding site -140 mediates this loss of TbetaRII expression in prostate cancer. METHODS: Sixty-seven prostate cancer (PC) samples, 8 benign prostatic hyperplasia (BPH) samples, and 4 prostate cancer cell lines (DUPro, LNCaP, ND-1 and PC-3) were analyzed for 1) TbetaRII mRNA expression by semiquantitative RT-PCR, 2) TbetaRII protein expression by immunohistochemistry, and 3) TGFbetaRII promoter methylation at CpG site -140 by methylation specific PCR and bisulfite DNA sequencing. Prostate cancer cell lines were treated with the demethylating agent 5aza2'deoxycytidine to determine if TbetaRII gene expression could be increased by blocking promoter methylation. RESULTS: mRNA and protein expression of TbetaRII was lower in the PC samples than in the BPH samples. CpG methylation at site -140 was higher in PC than in BPH (P < 0.01). Promoter methylation was inversely correlated with TbetaRII mRNA expression in the PC and BPH samples (P < 0.0001). PC3, ND1, and DUPro TbetaRII mRNA expression increased following treatment of cells with 5-aza-2'-deoxycytidine. CONCLUSION: CpG methylation of the TbetaRII promoter at CPG site -140 leads to functional loss of the TbetaRII gene in prostate cancer. Treatment with 5-aza-2' deoxycytidine can restore gene expression. The current study results report the first association between prostate cancer and loss of the TGF- beta signaling pathway by TbetaRII DNA promoter methylation.     

The loss of NKX3.1 expression in testicular--and prostate--cancers is not caused by promoter hypermethylation

BACKGROUND: Recent studies have demonstrated that the NKX3.1 protein is commonly down-regulated in testicular germ cell tumors (TGCTs) and prostate carcinomas. The homeobox gene NKX3.1 maps to chromosome band 8p21, which is a region frequently lost in prostate cancer, but not in TGCT. Mutations have not been reported in the NKX3.1 sequence, and the gene is hypothesized to be epigenetically inactivated. In the present study we examined the methylation status of the NKX3.1 promoter in relevant primary tumors and cell lines: primary TGCTs (n = 55), intratubular germ cell neoplasias (n = 7), germ cell tumor cell lines (n = 3), primary prostate adenocarcinomas (n = 20), and prostate cancer cell lines (n = 3) by methylation-specific PCR and bisulphite sequencing. RESULTS AND CONCLUSIONS: Down-regulation of NKX3.1 expression was generally not caused by promoter hypermethylation, which was only found in one TGCT. However, other epigenetic mechanisms, such as modulation of chromatin structure or modifications of histones, may explain the lack of NKX3.1 expression, which is seen in most TGCTs and prostate cancer specimens.     

Promoter hypermethylation and silencing of CHFR mitotic stress checkpoint gene in human gastric cancers

CHFR is a recently identified mitotic stress check-point gene. CHFR is ubiquitously expressed in normal human tissues, whereas loss of CHFR expression has been observed in human tumors. Silencing of CHFR has been associated with aberrant promoter methylation and histone deacetylation in several cancer types. In this study, we investigated epigenetic CHFR inactivation in human gastric cancers by examining CHFR expression and methylation status in gastric cancer cell lines with RT-PCR analysis, bisulfite PCR and sequencing. A series of primary gastric tumors were also analyzed for CHFR methylation. Eight of 12 (66.7%) gastric cancer cell lines and 19/43 (44.2%) primary gastric tumors showed CHFR methylation. In addition, CpG methylation status correlated well with CHFR expression in the human gastric cancer cell lines, in which treatment with 5-aza-dC resulted in de novo or enhanced expression of CHFR. Combination treatment of 5-aza-dC with trichostatin A showed a synergistic effect on CHFR expression in some cases. Our results indicate that aberrant promoter methylation of the CHFR gene was observed in a significant proportion of human gastric cancers and was responsible for the inactivation of the CHFR gene in gastric cancers.     

Detailed methylation analysis of the glutathione S-transferase pi (GSTP1) gene in prostate cancer

Glutathione-S-Transferases (GSTs) comprise a family of isoenzymes that provide protection to mammalian cells against electrophilic metabolites of carcinogens and reactive oxygen species. Previous studies have shown that the CpG-rich promoter region of the pi-class gene GSTP1 is methylated at single restriction sites in the majority of prostate cancers. In order to understand the nature of abnormal methylation of the GSTP1 gene in prostate cancer we undertook a detailed analysis of methylation at 131 CpG sites spanning the promoter and body of the gene. Our results show that DNA methylation is not confined to specific CpG sites in the promoter region of the GSTP1 gene but is extensive throughout the CpG island in prostate cancer cells. Furthermore we found that both alleles are abnormally methylated in this region. In normal prostate tissue, the entire CpG island was unmethylated, but extensive methylation was found outside the island in the body of the gene. Loss of GSTP1 expression correlated with DNA methylation of the CpG island in both prostate cancer cell lines and cancer tissues whereas methylation outside the CpG island in normal prostate tissue appeared to have no effect on gene expression.     

鼻咽癌细胞系中LTF基因的表达及遗传学与表观遗传学改变

目的 检测鼻咽癌(NPC)细胞系中LTF基因的表达水平,并分析遗传学和表观遗传学改变与其表达的关系.方法 采用逆转录聚合酶链反应(RT-PCR),检测NPC细胞系HNE1、HNE2、HNE3、CNE1、CNE2、5-8F、6-10B和15例慢性鼻咽炎组织中LTF mRNA表达水平,同时通过Western blot法分析6-10B细胞中LTF蛋白水平.运用微卫星分析技术、聚合酶链式反应-单链构象多态(PCR-SSCP)方法和甲基化特异性PCR(MSP)以及亚硫酸盐克隆测序方法,分别检测LTF的杂合性丢失、突变和启动子区甲基化情况.结果 在15例慢性鼻咽炎组织中,LTF基因稳定表达.在NPC细胞系中,除6-10B细胞有LTF基因弱表达外,其余均表达缺失,显著低于慢性鼻咽炎组织中的表达(Z=-3.738,P=0.000),且6-10B细胞中无LTF蛋白的表达.杂合性丢失分析显示,NPC细胞系中不存在LTF微卫星位点的缺失.突变检测显示,LTF在NPC细胞系中的突变率为14.3%(1/7),且突变存在于HNE1细胞系中,即启动子序列-305至-50 bp发生了缺失突变(-218delT).甲基化分析显示,LTF基因在慢性鼻咽炎组织中无甲基化,而在所有NPC细胞系中均有甲基化.用5-杂氮-2-脱氧包苷处理5-8F和6-10B细胞后,LTF mRNA表达上调.结论 LTF基因在NPC细胞系中表达失活,其分子机制与启动子高甲基化和缺失突变有关.     

Aberrant methylation of the Human Hedgehog interacting protein (HHIP) gene in pancreatic neoplasms

Hedgehog pathway overactivity has been implicated in the development of a variety of human cancers. The Human Hedgehog interacting protein (HHIP), a negative regulator of hedgehog signaling, has been shown to be underexpressed in pancreatic cancers. In this study we determined if the HHIP gene is a target for genetic and epigenetic alterations. While no mutations of HHIP were identified, we found complete methylation of the HHIP promoter CpG island in three pancreatic cancer cell lines, and partial hypermethylation in 13/17 (80%) pancreatic cancer cell lines, 35/75 (46%) primary pancreatic cancers and 14/18 (78%) pancreatic cancer xenografts, but no methylation in 13 normal pancreata.In pancreatic cancer cell lines, complete methylation was associated with absent or reduced HHIP expression by real-time RT-PCR. HHIP expression could be restored in methylated cell lines using epigenetic modifier drugs. Restoring the expression of HHIP in pancreatic cancer cells by 5-aza-2'-deoxycytidine led to a decrease in Gli reporter activity, consistent with downregulation of Hedgehog signaling. These results indicate in some pancreatic adenocarcinomas that HHIP is epigenetically inactivated by promoter methylation, and its silencing could contribute to the increased Hedgehog signaling observed in pancreatic neoplasms.     

Epigenetic suppression of secreted frizzled related protein 1 (SFRP1) expression in human breast cancer

Expression of Secreted frizzled related protein 1 (SFRP1), a recently identified tumor suppressor gene encoding a WNT signaling antagonist, has been found to be frequently down-regulated in breast cancer and is associated with disease progression and poor prognosis. Here, we investigated the role of epigenetic silencing of SFRP1 in breast cancer cell lines and primary breast tumors. Through analyses by methylation-specific PCR and bisulfite sequencing, promoter methylation of SFRP1 was detected in 88% (7/8) of breast cancer cell lines, 17% (1/6) of grade 1 of ductal carcinoma in situ (DCIS), 69% (9/13) of grade 2 and 3 of DCIS, 68% (19/28) of invasive ductal carcinomas (IDC) and 33% (6/18) of lobular carcinomas but not in any (0/14) of normal mammoplasty specimens and mammary epithelial organoids examined. Real-time RT-PCR studies indicated that loss or downregulation of SFRP1 expression in tumors is frequently associated with promoter hypermethylation. In addition, breast cancer cell lines with SFRP1 promoter hypermethylation reexpressed SFRP1 mRNA after treatment with 5-azaC, implying that DNA methylation is the predominant epigenetic mechanism for SFRP1 gene silencing. These findings suggest that frequent downregulation of SFRP1 expression in breast cancer can be attributed, in large part, to aberrant promoter hypermethylation in conjunction with or without histone deacetylation. Based on the frequency of tumor-specific hypermethylation in this gene, SFRP1 could provide a valuable marker for breast cancer.