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.     

Abberant methylation of p16, HIC1, N33 and GSTP1 genes in tumor epitelium and tumor-associated stromal cells of prostate cancer

The methylation status of four genes significant in prostate carcinogenesis p16, HIC1, N33 and GSTP1, were evaluated using quantitative methylationsensitive polymerase chain reaction. Tumor epithelia, tumor-associated stroma, normal epithelia, foci of PIN and benign prostate hyperplasia, and stroma adjacent to tumor tissues were isolated from whole-mount prostatectomy specimens of patients with localized prostate cancer by using laser capture microdissection. We found high levels of gene methylation in the tumor epithelium and tumor-associated stromal cells and some methylation in both hyperplastic epithelium and stromal cells in normal-appearing tissues located adjacent to tumors. Promoter methylation in the non-neoplastic cells of the prostate tumor microenvironment may play an important role in cancer development and progression. We examined the promoter methylation status of pl6, HIC1, N33 and GSTP1 in prostate biopsy fragments and prostate tissues after radical prostatectomy from patients with adenocarcinoma without laser capture microdissection. Methylation frequencies of all genes in tumor samples were considerably lower than frequencies in microdissected tumour samples (HIC1, 71 versus 89%; p16, 22 versus 78%; GSTP1, 32 versus 100%; N33, 20 versus 33%). The laser capture microdissection is required procedure in methylation studies taking into account multifocality and heterogenity of prostate cancer tissue.     

GSTP1 CpG island hypermethylation as a molecular biomarker for prostate cancer

Somatic hypermethylation of CpG island sequences at GSTP1, the gene encoding the pi-class glutathione S-transferase, appears to be characteristic of human prostatic carcinogenesis. To consider the potential utility of this epigenetic alteration as a biomarker for prostate cancer, we present here a comprehensive review of the literature describing somatic GSTP1 changes in DNA from prostate cells and tissues. GSTP1 CpG island hypermethylation has been detected in prostate cancer DNA using a variety of assay techniques, including (i) Southern blot analysis (SB), after treatment with (5-m)C-sensitive restriction endonucleases, (ii) the polymerase chain reaction, following treatment with (5-m)C-sensitive restriction endonucleases (RE-PCR), (iii) bisulfite genomic sequencing (BGS), and (iv) bisulfite modification followed by the polymerase chain reaction, using primers selective for target sequences containing (5-m)C (MSP). In the majority of the case series so far reported, GSTP1 CpG island hypermethylation was present in DNA from at least 90% of prostate cancer cases. When analyses have been carefully conducted, GSTP1 CpG island hypermethylation has not been found in DNA from normal prostate tissues, or from benign prostatic hyperplasia (BPH) tissues, though GSTP1 CpG island hypermethylation changes have been detected in DNA from candidate prostate cancer precursor lesions proliferative inflammatory atrophy (PIA) and prostatic intraepithelial neoplasia (PIN). Using PCR methods, GSTP1 CpG island hypermethylation has also been detected in urine, ejaculate, and plasma from men with prostate cancer. GSTP1 CpG island hypermethylation, a somatic epigenetic alteration, appears poised to serve as a molecular biomarker useful for prostate cancer screening, detection, and diagnosis.     

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.     

DNA甲基化异常与肿瘤耐药

肿瘤耐药是导致肿瘤化疗失败的主要原因, 其产生机制复杂多样, 是多种因素共同作用的结果。近年来, 表观遗传改变在肿瘤耐药中的作用日益受到关注。DNA甲基化是一种重要的表观遗传修饰, 在调节基因表达和维持基因组稳定性中扮演着重要角色。原发性或获得性耐药的肿瘤细胞大多伴随DNA异常甲基化, 越来越多的证据显示, DNA甲基化异常是肿瘤细胞耐药表型产生的重要机制。文章就DNA甲基化异常与肿瘤细胞耐药的关系及相关作用机制进行了综述。     

Sex steroid-induced DNA methylation changes and inflammation response in prostate cancer

BackgroundSex steroid hormones have been reported to induce inflammation causing dysregulation of cytokines in prostate cancer cells. However, the underlying epigenetic mechanism has not well been studied. The objective of this study was to evaluate the effect of sex steroid hormones on epigenetic DNA methylation changes in prostate cancer cells using a signature PCR methylation array panel that correspond to 96 genes with biological function in the human inflammatory and autoimmune signals in prostate cancer. Of the 96-gene panel, 32 genes showed at least 10% differentially methylation level in response to hormonal treatment when compared to untreated cells. Genes that were hypomethylated included CXCL12, CXCL5, CCL25, IL1F8, IL13RAI, STAT5A, CXCR4 and TLR5; and genes that were hypermethylated included ELA2, TOLLIP, LAG3, CD276 and MALT1. Quantitative RT-PCR analysis of select genes represented in a cytokine expression array panel showed inverse association between DNA methylation and gene expression for TOLLIP, CXCL5, CCL18 and IL5 genes and treatment of prostate cancer cells with 5′-aza-2′-deoxycytidine with or without trichostatin A induced up-regulation of TOLLIP expression. Further analysis of relative gene expression of matched prostate cancer tissues when compared to benign tissues from individual patients with prostate cancer showed increased and significant expression for CCL18 (2.6-fold; p < 0.001), a modest yet significant increase in IL5 expression (1.17-fold; p = 0.015), and a modest increase in CXCL5 expression (1.4-fold; p = 0.25). In conclusion, our studies demonstrate that sex steroid hormones can induce aberrant gene expression via differential methylation changes in prostate carcinogenesis.     

MicroRNA-487a-3p functions as a new tumor suppressor in prostate cancer by targeting CCND1

Prostate cancer (PCa) is one of the major health problems of the aging male. The roles of dysregulated microRNAs in PCa remain unclear. In this study, we mined the public published data and found that miR-487a-3p was significantly downregulated in 38 pairs of clinical prostate tumor tissues compared with the normal tissues. We further verified this result by in situ hybridization on tissue chip and quantitative real-time polymerase chain reaction (qRT-PCR) in PCa/normal cells. miR-487a-3p targeting of cyclin D1 (CCND1) was identified using bioinformatics, qRT-PCR and western blot analyses. The cellular proliferation, cell cycle, migration, and invasion were assessed by cell counting kit-8, flow cytometry analysis and transwell assay. We discovered that overexpression of miR-487a-3p suppressed PCa cell growth, migration, invasion by directly targeting CCND1. Knockdown of CCND1 in PCa cells showed similar results. Meanwhile, the expression level of CCND1 was significantly upregulated in the PCa tissues and cell lines, which presented negative correlation with the expression of miR-487a-3p. More important, we demonstrated significantly reduced growth of xenograft tumors of stable miR-487a-3p-overexpressed human PCa cells in nude mice. Taken together, for the first time, our results revealed that miR-487a-3p as a tumor suppressor of PCa could target CCND1. Our finding might reveal miR-487a-3p could be potentially contributed to the pathogenesis and a clinical biomarker or the new potential therapeutic target of PCa.     

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.     

Aberrant Keap1 methylation in breast cancer and association with clinicopathological features

Keap1 (Kelch-like ECH-associated protein 1) is an adaptor protein that mediates the ubiquitination/degradation of genes regulating cell survival and apoptosis under oxidative stress conditions. We determined methylation status of the KEAP1 promoter in 102 primary breast cancers, 14 pre-invasive lesions, 38 paired normal breast tissues and 6 normal breast from reductive mammoplasty by quantitative methylation specific PCR (QMSP). Aberrant promoter methylation was detected in 52 out of the 102 primary breast cancer cases (51%) and 10 out of 14 pre-invasive lesions (71%). No mutations of the KEAP1 gene were identified in the 20 breast cancer cases analyzed by fluorescence based direct sequencing. Methylation was more frequent in the subgroup of patients identified as ER positive-HER2 negative tumors (66.7%) as compared with triple-negative breast cancers (35%) (p = 0.05, Chi-square test). The impact of the interactions between Er, PgR, Her2 expression and KEAP1 methylation on mortality was investigated by RECPAM multivariable statistical analysis, identifying four prognostic classes at different mortality risks. Triple-negative breast cancer patients with KEAP1 methylation had higher mortality risk than patients without triple-negative breast cancer (HR = 14.73, 95%CI: 3.65–59.37). Both univariable and multivariable COX regressions analyses showed that KEAP1 methylation was associated with a better progression free survival in patients treated with epirubicin/cyclophosfamide and docetaxel as sequential chemotherapy (HR = 0.082; 95%CI: 0.007–0.934). These results indicate that aberrant promoter methylation of the KEAP1 gene is involved in breast cancerogenesis. In addition, identifying patients with KEAP1 epigenetic abnormalities may contribute to disease progression prediction in breast cancer patients.     

Aberrant methylation of tumor suppressor genes and allelic imbalance in cervical intraepithelial neoplasia

The methylation status of genes p16, MLH1, HIC1, MGMT, N33, and RB1 was determined in cervical smears of women without gynecological pathology, in biopsies from patients with high-grade cervical intraepithelial neoplasia (CIN3), and in samples of nondysplastic cervical tissue adjacent to CIn3. The level of methylation of these genes in normal smears was insignificant. In CIN3, methylation frequencies were as follows: 58% for p16, 51% for MLH1, 84% for HIC1, and 27% for N33. However, dysplastic tissues did not differ significantly from the control with respect to the methylation frequencies of the MGMT and RB1 genes (8 and 15%, respectively). The methylation frequency of the suppressor genes in nondysplastic, morphologically normal adjacent tissues was also high. We found that 21% of samples (9/42) had microsatellite instability at chromosomes 5q11.2–q14.3 (3/42) and 13q14–q14.3 (6/42). Loss of heterozygosity (LOH) at region 13q14 was detected in 7% of samples (3/42).     

Zebularine partially reverses GST methylation in prostate cancer cells and restores sensitivity to the DNA minor groove binder brostallicin

Brostallicin is a DNA minor groove binder that shows enhanced antitumor activity in cells with high glutathione S-transferase (GST)/glutathione content. Prostate cancer cells present, almost invariably, methylation of the GSTP1 gene promoter and, as a consequence, low levels of GST-pi expression and activity. In these cells, brostallicin shows very little activity. We tested whether pretreatment of heavily GST-methylated prostate cancer cells with demethylating agents could enhance the activity of brostallicin. Human prostate cancer cells LNCaP and DU145 were used for these studies both in vitro and in vivo. The demethylating agent zebularine was used in combination with brostallicin. Methylation specific PCR and pyrosequencing were used to determine the level of GST methylation. Pretreatment with demethylating agents enhanced the in vitro activity of brostallicin in LNCaP cells. Zebularine, in particular, induced an enhancement of activity in vivo comparable to that obtained by transfecting the human GSTP1 gene in LNCaP cells in vitro. Molecular analysis performed on tumor xenografts in mice pretreated with zebularine failed to detect re-expression of GST-pi and demethylation of GSTP1. However, we found demethylation in the GSTM1 gene, with consequent re-expression of GST-mu at the mRNA level. These results indicate that zebularine, both in vitro and in vivo, enhances the activity of brostallicin and that this enhancement correlates with re-expression of GST-pi and GST-mu. These findings highlight the potential therapeutic value of combining demethylating agents and brostallicin in tumors with GST methylation that poorly respond to brostallicin.     

Aberrant p63 and WT-1 expression in myoepithelial cells of pregnancy-associated breast cancer: implications for tumor aggressiveness and invasiveness

Our recent studies revealed that focal alterations in breast myoepithelial cell layers significantly impact the biological presentation of associated epithelial cells. As pregnancy-associated breast cancer (PABC) has a significantly more aggressive clinical course and mortality rate than other forms of breast malignancies, our current study compared tumor suppressor expression in myoepithelial cells of PABC and non-PABC, to determine whether myoepithelial cells of PABC may have aberrant expression of tumor suppressors. Tissue sections from 20 cases of PABC and 20 cases of stage, grade, and age matched non-PABC were subjected to immunohistochemistry, and the expression of tumor suppressor maspin, p63, and Wilms' tumor 1 (WT-1) in calponin positive myoepithelial cells were statistically compared. The expression profiles of maspin, p63, and WT-1 in myoepithelial cells of all ducts encountered were similar between PABC and non-PABC. PABC, however, displayed several unique alterations in terminal duct and lobular units (TDLU), acini, and associated tumor tissues that were not seen in those of non-PABC, which included the absence of p63 and WT-1 expression in a vast majority of the myoepithelial cells, cytoplasmic localization of p63 in the entire epithelial cell population of some lobules, and substantially increasing WT-1 expression in vascular structures of the invasive cancer component. All or nearly all epithelial cells with aberrant p63 and WT-1 expression lacked the expression of estrogen receptor and progesterone receptor, whereas they had a substantially higher proliferation index than their counterparts with p63 and WT-1 expression. Hyperplastic cells with cytoplasmic p63 expression often adjacent to, and share a similar immunohistochemical and cytological profile with, invasive cancer cells. To our best knowledge, our main finings have not been previously reported. Our findings suggest that the functional status of myoepithelial cells may be significantly associated with tumor aggressiveness and invasiveness.     

Angiogenesis and prostate cancer tumor growth

The initiation of new blood vessels through angiogenesis is critical to tumor growth. Tumor cells release soluble angiogenic factors that induce neovascularization, without which nutrients and oxygen would not be available to allow tumors to grow more than 2-3 mm in diameter. This "angiogenic switch" or angiogenic phenotype requires an imbalance between proangiogenic and antiangiogenic factors since the formation of new blood vessels is highly regulated. This review discusses angiogenesis mediators, and the potential for manipulation of angiogenic factors as a practical cancer therapy, particularly in prostate cancer.     

ING5 inhibits cancer aggressiveness by inhibiting Akt and activating p53 in prostate cancer

Prostate cancer (PCa) is one of the most common types of cancer in men. In several recent studies, chromosomal deletions in the q arm of chromosome 2, where ING5 resides within, have been identified in various cancer types including PCa. In this study, we investigate the role of ING5 as a tumor suppressor in PCa. We examined the expression level of ING5 in tissue samples and cell lines using quantitative real‐time polymerase chain reaction and western blot analysis. We tested the in vitro tumor suppressor potential of ING5 in PC3 and LNCaP cells stably overexpressing it using cell viability, colony formation, migration, invasion, and apoptosis assays. We then investigated the effects of ING5 on the Akt and p53 signaling using western blot analysis. We show that ING5 is significantly downregulated in PCa tumor tissue samples and cell lines compared with the corresponding controls. In vitro assays demonstrate that ING5 effectively suppresses proliferative, clonogenic, migratory, and invasive potential and induce apoptosis in PCa cells. ING5 may potentially exert its anti‐tumor potential by inhibiting AKT and inducing p53 signaling pathways. Our findings demonstrate that ING5 possesses tumor suppressor roles in vitro, pointing its importance during the prostatic carcinogenesis processes.     

Induction of apoptosis in prostate tumor PC-3 cells and inhibition of xenograft prostate tumor growth by the vanilloid capsaicin

Capsaicin, the pungent ingredient of hot chilli pepper, has been recently shown to induce apoptosis in several cell lines through a not well known mechanism. Here, we investigated the role of the vanilloid capsaicin in the death regulation of the human cancer androgen-resistant cell line PC-3. Capsaicin inhibited the growth of PC-3 with an IC₅₀ of 20 μM cells and induced cell apoptosis, as assessed by flow cytometry and nuclei staining with DAPI. Capsaicin induced apoptosis in prostate cells by a mechanism involving reactive oxygen species generation, dissipation of the mitochondrial inner transmembrane potential (ΔΨ_m) and activation of caspase 3. Capsaicin-induced apoptosis was not reduced by the antagonist capsazepine in a dose range from 0.1 μM to 20 μM, suggesting a receptor-independent mechanism. To study the in vivo effects of capsaicinoids, PC-3 cells were grown as xenografts in nude mice. Subcutaneous injection of either capsaicin or capsazepine (5 mg/kg body weight) in nude mice suppressed PC-3 tumor growth in all tumors investigated and induced apoptosis of tumor cells. Our data show a role for capsaicin against androgen-independent prostate cancer cells in vitro and in vivo and suggest that capsaicin is a promising anti-tumor agent in hormone-refractory prostate cancer, which shows resistance to many chemotherapeutic agents.     

CDO1 promoter methylation is associated with gene silencing and is a prognostic biomarker for biochemical recurrence-free survival in prostate cancer patients

Molecular biomarkers may facilitate the distinction between aggressive and clinically insignificant prostate cancer (PCa), thereby potentially aiding individualized treatment. We analyzed cysteine dioxygenase 1 (CDO1) promoter methylation and mRNA expression in order to evaluate its potential as prognostic biomarker. CDO1 methylation and mRNA expression were determined in cell lines and formalin-fixed paraffin-embedded prostatectomy specimens from a first cohort of 300 PCa patients using methylation-specific qPCR and qRT-PCR. Univariate and multivariate Cox proportional hazards and Kaplan-Meier analyses were performed to evaluate biochemical recurrence (BCR)-free survival. Results were confirmed in an independent second cohort comprising 498 PCa cases. Methylation and mRNA expression data from the second cohort were generated by The Cancer Genome Atlas (TCGA) Research Network by means of Infinium HumanMethylation450 BeadChip and RNASeq. CDO1 was hypermethylated in PCa compared to normal adjacent tissues and benign prostatic hyperplasia (P < 0.001) and was associated with reduced gene expression (ρ = ?0.91, P = 0.005). Using two different methodologies for methylation quantification, high CDO1 methylation as continuous variable was associated with BCR in univariate analysis (first cohort: HR = 1.02, P = 0.002, 95% CI [1.01–1.03]; second cohort: HR = 1.02, P = 0.032, 95% CI [1.00–1.03]) but failed to reach statistical significance in multivariate analysis. CDO1 promoter methylation is involved in gene regulation and is a potential prognostic biomarker for BCR-free survival in PCa patients following radical prostatectomy. Further studies are needed to validate CDO1 methylation assays and to evaluate the clinical utility of CDO1 methylation for the management of PCa.