Aberrant methylation of p16, HIC1, N33, and GSTP1 in tumor epithelium and tumor-associated cells in prostate cancer

The methylation status of p16, HIC1, N33, and GSTP1, which are involved in prostate carcinogenesis, was studied in prostate tissue samples containing neoplasms. Malignant acini, prostatic intraepithelial neoplasia (PIN) and benign prostatic hyperplasia (BPH) foci, and stroma surrounding glandular structures of each type were detected in histological sections, using laser capture microdissection of prostate tissue. High levels of methylation were found in tumor epithelium and adjacent tumor-associated stromal cells. Epigenetic changes in the stroma are indicative of a major role of tumor microenvironment in cancer development and progression. The methylation status of p16, HIC1, N33, and GSTP1 was also assessed in prostate biopsy material and operative tumor samples without laser capture microdissection. The methylation frequencies of all genes in tumor samples were considerably lower than those in microdissected tumor samples (HIC1, 71% vs. 89%; p16, 22% vs. 78%; GSTP1, 32% vs. 100%; and N33, 20% vs. 33%, respectively). It was concluded that laser capture micro-dissection is required in molecular analysis of tumors of this type.     

Frequent allelic losses in tumor-associated stromal cells and tumor epitelium of prostate cancer

It has become increasingly clear that tumor microenvironment plays a critical role in carcinogenesis. Accumulation of genetic alterations is typical not only for cancer epithelial cells but tumor-associated fibroblasts as well. Tumor epithelia, tumor-associated stroma from prostatectomy specimens of patients with prostate cancer and cells from prostatic intraepithelial neoplasia (PIN) and adjacent stroma from males with PIN were isolated by using laser capture microdissection. Microsatellite allelotyping was evaluated using 4 highly polymorphic markers for chromosomal regions 8p22, 16q23-24 and 13q14. Incidences of alterations (loss of heterozygosity or allelic imbalance) were 48% for region 8p22, 72% for 16q23 and 37% for 13q14. The LOH frequencies in tumor-associated stroma cells were very similar. Alterations at chromosome 13q were significantly associated with advanced tumor stage, whereas AI at 16q was also associated with high Gleason score and lymph node metastasis. We find some incidences of allelic imbalance in premalignant lesions in epithelial (16-27%) and stromal (7-22%) components. Our results show that the frequencies of genetic aberrations are as high in stromal cells as in tumor cells.     

Alterations in promoter methylation status of tumor suppressor HIC1, SFRP2, and DAPK1 genes in prostate carcinomas

Hypermethylated genomic DNA is a common feature in tumoral tissues, although the prevalence of this modification remains poorly understood. We aimed to determine the frequency of five tumor suppressor (TS) genes in prostate cancer and the correlation between promoter hypermethylation of these genes and low and high grade of prostate carcinomas. A total of 30 prostate tumor specimens were investigated for promoter methylation status of TS hypermethylated in cancer 1 (HIC1), death-associated protein kinase 1 (DAPK1), secreted frizzled-related protein 2 (SFRP2), cyclin-dependent kinase inhibitor 2A (p16), and O-6-methylguanine-DNA methyltransferase (MGMT) genes by using bisulfite modifying method. A high frequency of promoter hypermethylation was found in HIC1 (70.9%), SFRP2 (58.3%), and DAPK1 (33.3%) genes in tumor samples that were examined. The current data show high frequency of hypermethylation changes in HIC1, SFRP2, and DAPK1 genes in prostate carcinomas of high Gleason Score (GS).     

Loss of heterozygosity and microsatellite instability in tumor-associated stromal cells and tumor epithelium of prostate cancer

In the past decade, intense studies of the tumor microenvironment yielded ample data testifying to the critical role of stroma in carcinogenesis. Genetic lesions accumulate not only in the tumor epithelium, but also in tumor-associated fibroblasts. The epithelial and stromal components of prostate cancer (PC) and prostatic intraepithelial neoplasia (PIN) were isolated by laser capture microdissection and subjected to microsatellite analysis of chromosome regions 8p22, 13q14, and 16q23. The frequency of allele alterations in the epithelium was 48% for 8p22, 72% for 13q14, and 37% for 16q23. Slightly higher frequencies of the loss of heterozygosity (LOH) and microsatellite instability in these loci were observed in tumor-associated stroma. Molecular alterations were also found in both epithelial (16–27%) and stromal (8–22%) components in PIN. LOH on chromosomes 16 and 13 in the epithelium was significantly associated with the Gleason score, PC stage, and metastasis into regional lymph nodes. Thus, multiple genetic aberrations occur in the stromal component of PC as frequently as in the tumor epithelium.     

GSTP1 DNA methylation and expression status is indicative of 5-aza-2'-deoxycytidine efficacy in human prostate cancer cells

DNA methylation plays an important role in carcinogenesis and the reversibility of this epigenetic modification makes it a potential therapeutic target. To date, DNA methyltransferase inhibitors (DNMTi) have not demonstrated clinical efficacy in prostate cancer, with one of the major obstacles being the inability to monitor drug activity during the trial. Given the high frequency and specificity of GSTP1 DNA methylation in prostate cancer, we investigated whether GSTP1 is a useful marker of DNMTi treatment efficacy. LNCaP prostate cancer cells were treated with 5-aza-2'-deoxycytidine (5-aza-CdR) either with a single high dose (5-20 μM), every alternate day (0.1-10 μM) or daily (0.005-2.5 μM). A daily treatment regimen with 5-aza-CdR was optimal, with significant suppression of cell proliferation achieved with doses of 0.05 μM or greater (p<0.0001) and induction of cell death from 0.5 μM (p<0.0001). In contrast, treatment with a single high dose of 20 μM 5-aza-CdR inhibited cell proliferation but was not able to induce cell death. Demethylation of GSTP1 was observed with doses of 5-aza-CdR that induced significant suppression of cell proliferation (≥ 0.05 μM). Re-expression of the GSTP1 protein was observed only at doses of 5-aza-CdR (≥ 0.5 μM) associated with induction of cell death. Treatment of LNCaP cells with a more stable DNMTi, Zebularine required at least a 100-fold higher dose (≥ 50 μM) to inhibit proliferation and was less potent in inducing cell death, which corresponded to a lack of GSTP1 protein re-expression. We have shown that GSTP1 DNA methylation and protein expression status is correlated with DNMTi treatment response in prostate cancer cells. Since GSTP1 is methylated in nearly all prostate cancers, our results warrant its testing as a marker of epigenetic therapy response in future clinical trials. We conclude that the DNA methylation and protein expression status of GSTP1 are good indicators of DNMTi efficacy.     

GCPII modulates oxidative stress and prostate cancer susceptibility through changes in methylation of RASSF1, BNIP3, GSTP1 and Ec-SOD

Glutamate carboxypeptidase II (GCPII) haplotypes were found to influence susceptibility to prostate cancer. In the current study, we have elucidated the impact of these haplotypes on the expression of PSMA, BNIP3, Ec-SOD, GSTP1 and RASSF1 genes to understand the epigenetic basis of oxidative stress and prostate cancer risk. Expression analysis was carried out by RT-PCR. Bisulphite treated DNA was subjected to MS-PCR and COBRA for epigenetic studies. Plasma MDA and glutathione levels were measured. In prostate cancer, upregulation of BNIP3 (204.4 ± 23.77 vs. 143.9 ± 16.42 %, p = 0.03); and downregulation of Ec-SOD (105.8 ± 13.69 vs. 176.3 ± 21.1 %, p = 0.027) and RASSF1A (16.67 ± 16.0 vs. 90.8 ± 8.5 %, p = 0.0048) was observed. Hypomethylation of BNIP3 (31.25 ± 16.19 vs. 45.70 ± 2.42 %, p < 0.0001), hypermethylation of Ec-SOD (71.4 ± 6.75 vs. 10.0 ± 3.78 %, p < 0.0001) and RASSF1 (76.25 ± 12.53 vs. 30.0 ± 8.82 %, p = 0.0077) was observed in prostate cancer. The gene expression signature of PSMA, BNIP3, Ec-SOD, GSTP1, clearly demarcated cases and controls (AUC = 0.89 in the ROC curve). D191V variant of GCPII showed positive association with oxidative stress and inverse association with Ec-SOD expression. H475Y variant showed positive association with Ec-SOD expression and inverse association with oxidative stress. R190W variant was found to reduce oxidative stress by increasing glutathione levels. GCPII genetic variants contribute to increased oxidative stress and prostate cancer risk by modulating the CpG island methylation of Ec-SOD.     

Bone marrow-derived EP3-expressing stromal cells enhance tumor-associated angiogenesis and tumor growth

Recent results suggest that bone marrow (BM)-derived hematopoietic cells are major components of tumor stroma and play crucial roles in tumor growth and angiogenesis. An E-type prostaglandin is known to regulate angiogenesis. We examined the role of BM-derived cells expressing an E-type prostaglandin receptor subtype (EP3) in tumor-induced angiogenesis and tumor growth. The replacement of wild-type (WT) BM with BM cells (BMCs) from green fluorescent protein (GFP) transgenic mice revealed that the stroma developed via the recruitment of BMCs. Selective knockdown of EP3 by recruitment of genetically modified BMCs lacking EP3 receptors was performed by transplantation of BMCs from EP3 knockout (EP3^−/−) mice. Tumor growth and tumor-associated angiogenesis were suppressed in WT mice transplanted with BMCs from EP3^−/− mice, but not in mice transplanted with BMCs from either EP1^−/−, EP2^−/−, or EP4^−/− mice. Immunohistochemical analysis revealed that vascular endothelial growth factor (VEGF) expression was suppressed in the stroma of mice transplanted with BMCs from EP3^−/− mice. EP3 signaling played a significant role in the recruitment of VEGFR-1- and VEGFR-2-positive cells from the BM to the stroma. These results indicate that the EP3 signaling expressed in bone marrow-derived cells has a crucial role in tumor-associated angiogenesis and tumor growth with upregulation of the expression of the host stromal VEGF together with the recruitment of VEGFR-1/VEGFR-2-positive. The present study suggests that the blockade of EP3 signaling and the recruitment of EP3-expressing stromal cells may become a novel strategy to treat solid tumors.     

Ischemia dysregulates DNA methyltransferases and p16INK4a methylation in human colorectal cancer cells

Epigenetic modifications are involved in the initiation and progression of cancer. Expression patterns and activity of DNA methyltransferases (DNMTs) are strictly controlled in normal cells, however, regulation of these enzymes is lost in cancer cells due to unknown reasons. Cancer therapies which target DNMTs are promising treatments of hematologic cancers, but they lack effectiveness in solid tumors. Solid tumors exhibit areas of hypoxia and hypoglycaemia due to their irregular and dysfunctional vasculature, and we previously showed that hypoxia reduces global DNA methylation. Colorectal carcinoma (CRC) cells (HCT116 and 379.2; p53+/+ and p53-/-, respectively) were subjected to ischemia (hypoxia and hypoglycaemia) in vitro, and levels of DNMTs were assessed. We found a significant decrease in mRNA for DNMT1, DNMT3a and DNMT3b, and similar reductions in DNMT1 and DNMT3a protein levels were detected by western blotting. In addition, total activity levels of DNMTs (as measured by an ELISA-based DNMT activity assay) were reduced in cells exposed to hypoxic and hypoglycaemic conditions. Immunofluorescence of HCT116 tumor xenografts demonstrated an inverse relationship between ischemia (as revealed by carbonic anhydrase IX staining) and DNMT1 protein. Bisulfite sequencing of the proximal promoter region of p16INK4a showed a decrease in 5-methylcytosine following in vitro exposure to ischemia. These studies provide evidence for the down-regulation of DNMTs and modulation of methylation patterns by hypoxia and hypoglycaemia in human CRC cells, both in vitro and in vivo. Our findings suggest that ischemia, either intrinsic or induced through the use of anti-angiogenic drugs, may influence epigenetic patterning and hence tumor progression.     

Ischemia dysregulates DNA methyltransferases and p16INK4a methylation in human colorectal cancer cells

Epigenetic modifications are involved in the initiation and progression of cancer. Expression patterns and activity of DNA methyltransferases (DNMTs) are strictly controlled in normal cells; however, regulation of these enzymes is lost in cancer cells due to unknown reasons. Cancer therapies which target DNMTs are promising treatments of hematologic cancers, but they lack effectiveness in solid tumors. Solid tumors exhibit areas of hypoxia and hypoglycaemia due to their irregular and dysfunctional vasculature, and we previously showed that hypoxia reduces global DNA methylation. Colorectal carcinoma (CRC) cells (HCT116 and 379.2; p53^+/+ and p53^-/-, respectively) were subjected to ischemia (hypoxia and hypoglycaemia) in vitro and levels of DNMTs were assessed. We found a significant decrease in mRNA for DNMT1, DNMT3a and DNMT3b, and similar reductions in DNMT1 and DNMT3a protein levels were detected by western blotting. In addition, total activity levels of DNMTs (as measured by an ELISA-based DNMT activity assay) were reduced in cells exposed to hypoxic and hypoglycaemic conditions. Immunofluorescence of HCT116 tumor xenografts demonstrated an inverse relationship between ischemia (as revealed by carbonic anhydrase IX staining) and DNMT1 protein. Bisulfite sequencing of the proximal promoter region of p16^INK4a showed a decrease in 5-methylcytosine following in vitro exposure to ischemia. These studies provide evidence for the downregulation of DNMTs and modulation of methylation patterns by hypoxia and hypoglycaemia in human CRC cells, both in vitro and in vivo. Our findings suggest that ischemia, either intrinsic or induced through the use of anti-angiogenic drugs, may influence epigenetic patterning and hence tumor progression.Key words: DNA methylation, DNA methyltransferases, colorectal carcinoma, ischemia, p53, hypoxia, hypoglycaemia     

Study of p16 promoter methylation in Egyptian colorectal cancer patients

Many tumor-suppressor genes contain CpG islands in their promoter regions which raised the necessity of investigating the role of methylation in silencing these genes. We examined p16 methylation as a potential biomarker in the peripheral blood of colorectal cancer (CRC) patients. Using methylation-specific polymerase chain reaction method, the methylation status of p16 was investigated in the tumor tissue and blood of 65 CRC patients and blood samples from 70 healthy control individuals. Also, the relationship between p16 methylation level and the clinical-pathological findings in CRC was evaluated. The frequency of blood p16 methylation in CRC cases was significantly higher than in control (P = 0.0001). The sensitivity and specificity of p16 methylation in diagnosing CRC was 55.38% and 98.5%, respectively, with 77.7% diagnostic accuracy. There was significant association between p16 methylation and age, sex, Dukes staging, lymph node involvement, and carcinoembryonic antigen levels. Our study revealed that p16 promoter methylation could be considered as both potential diagnostic and prognostic biomarker of CRC.     

Identification of novel subregions of LOH in gastric cancer and analysis of the HIC1 and TOB1 tumor suppressor genes in these subregions

Previously, we identified 3 overlapping regions showing loss of heterozygosity (LOH, R(1)-R(3) from 11 to 30 cM) on chromosome 17 in 45 primary gastric cancers (GCs). The data indicated the presence of tumor suppressor genes (TSGs) on chromosome 17 involved in GC. Among the putative TSGs in these regions, HIC1 (in SR(1)) and TOB1 (in SR(3)) remain to be examined in GC. By immunohistochemistry (IHC), methylation-specific PCR (MSP) and western blot, we evaluated the expression and regulation status for HIC1 and TOB1 protein in GC. We narrowed down the deletion intervals on chromosome 17 and defined five smaller LOH subregions, SR(1)-SR(5) (0.54 to 3.42 cM), in GC. We found that HIC1 had downregulated expression in 86% (91/106) and was methylated in 87% (26/30) of primary GCs. Of the primary GCs showing downregulation of HIC1 protein, 75% (18/24) had methylated HIC1 gene. TOB1 was either absent or expressed at reduced levels in 75% (73/97) of the GC samples. In addition, a general reduction was found in total and the ratio of unphosphorylated to phosphorylated TOB1 protein levels in the differentiated GC cell lines. Further analysis revealed significant simultaneous downregulation of both HIC1 and TOB1 protein in GC tissue microarray samples (67%, 52/78) and in primary GCs (65%, 11/17). These results indicate that silencing of HIC1 and TOB1 expression is a common occurrence in GC and may contribute to the development and progression of the disease.     

p53负调控前列腺癌细胞中PC-1基因的表达

在前列腺癌进展中发生的PC-1基因表达失调和p53基因突变,提示这两个事件之间可能存在的联系.用依托泊苷处理前列腺癌LNCaP细胞后,PC-1蛋白的表达受抑制;瞬时转染分析表明野生型p53负调控PC-1启动子的转录活性;缺失突变分析将PC-1基因启动子上受p53负调控的区域定位在翻译起始位点上游757 bp～323 bp之间.缺失PC-1启动子上的雄激素受体反应元件并没有消除p53对其转录活性的抑制作用;无论p53是否存在,组蛋白去乙酰化酶抑制剂TSA处理LNCaP细胞后可以导致PC-1启动子转录活性升高.因此,p53和去乙酰化酶可以独立抑制PC-1启动子活性.这些研究结果表明,野生型p53负调控PC-1基因启动子的转录活性,而前列腺癌进展过程中p53突变可能和PC-1基因的表达失调有关.     

Age-dependent methylation of ESR1 gene in prostate cancer

The incidence of prostate cancer increases dramatically with age and the mechanism underlying this association is unclear. Age-dependent methylation of estrogen receptor alpha (ESR1) gene has been previously implicated in other cancerous and benign diseases. We evaluated the age-dependent methylation of ESR1 in prostate cancer. The methylation status of ESR1 in 83 prostate cancer samples from patients aged 49 to 77 years (mean age at 67.4 years) was examined using the bisulfite genomic sequencing technique. The samples were divided into three age groups: men aged 60 years and under (n = 14), men aged 61-70 years (n = 40), and men aged over 70 years (n = 29). Overall, ESR1 promoter methylation was detected in 54 out of 83 (65.1%) prostate samples. The methylation rate of ESR1 increased dramatically with age from 50.0% in patients aged 60 years and under to 89.7% for patients aged 70 years and over. Logistic regression analyses revealed that age and Gleason score were the only variables that affect incidence of ESR1 methylation; other clinical factors such as prostate-specific antigen level and clinical stage did not. We also calculated ESR1 methylation density (the percentage of methylated CpGs among all CpGs within the analyzed region) and severity (the percentage of methylated CpG alleles) for each sample analyzed. Multiple regression analyses showed a positive correlation between age and methylation density (beta, 0.35; P, 0.012; 95% CI, 0.26-2.01); while Gleason score was positively associated with methylation severity (beta, 0.45; P, 0.018; 95% CI, 1.04-4.26). These findings suggest that methylation of ESR1 is both age-dependent and tumor differentiation-dependent and age-dependent methylation of ESR1 may represent a mechanism linking aging and prostate cancer.     

Abnormal methylation of several tumor suppressor genes in sporadic breast cancer

Multiplex methylation-sensitive PCR was employed in studying the methylation of CpG islands in the RB1, p16/CDKN2A, p15/CDKN2B, p14/ARF, CDH1, MGMT, HIC1, and N33 promoter regions in breast carcinoma (105 tumors). Methylation was often observed for the two major suppressor genes involved in cell-cycle control through the Cdk-Rb-E2F signaling pathway, RB1 (18/105, 17%) and p16 (59/105, 56%); both genes were methylated in 13 tumors. Methylation involved p15 in two (2%) tumors; CDH1, in 83 (79%) tumors; MGMT, in eight (8%) tumors, and N33, in nine (9%) tumors. The p14 promoter was not methylated in the tumors examined.     

LINE-1 methylation status in prostate cancer and non-neoplastic tissue adjacent to tumor in association with mortality

Aberrant DNA methylation seems to be associated with prostate cancer behavior. We investigated LINE-1 methylation in prostate cancer and non-neoplastic tissue adjacent to tumor (NTAT) in association with mortality from prostate cancer. We selected 157 prostate cancer patients with available NTAT from 2 cohorts of patients diagnosed between 1982–1988 and 1993–1996, followed up until 2010. An association between LINE-1 hypomethylation and prostate cancer mortality in tumor was suggested [hazard ratio per 5% decrease in LINE-1 methylation levels: 1.40, 95% confidence interval (CI): 0.95–2.01]. After stratification of the patients for Gleason score, the association was present only for those with a Gleason score of at least 8. Among these, low (<75%) vs. high (>80%) LINE-1 methylation was associated with a hazard ratio of 4.68 (95% CI: 1.03–21.34). LINE-1 methylation in the NTAT was not associated with prostate cancer mortality. Our results are consistent with the hypothesis that tumor tissue global hypomethylation may be a late event in prostate cancerogenesis and is associated with tumor progression.     

Lycopene and apo-10'-lycopenal do not alter DNA methylation of GSTP1 in LNCaP cells

DNA hypermethylation and silencing of tumor-suppressor genes are commonly seen in human cancers, and likely contribute to the process of carcinogenesis. A growing body of evidence suggests that dietary compounds may alter cancer risk through epigenetic modifications. Glutathione S-transferase P1 (GSTP1) is hypermethylated in >90% of prostate cancer cases making it one of the most common genome alterations in prostate cancer. LNCaP cells were treated either with lycopene or apo-10′-lycopenal for 7 days, and mRNA expression and DNA methylation of GSTP1 were evaluated. Neither compound significantly altered expression nor methylation of GSTP1 while treatment with 5-azacytidine decreased methylation by 78%. These findings demonstrate that lycopene and apo-10′-lycopenal are not effective demethylating agents of GSTP1 in the human LNCaP cell line.