Epigenetic suppression of the anti-aging gene KLOTHO in human prostate cancer cell lines

KLOTHO was originally identified as an aging-suppressor gene that causes a human aging-like phenotype when tested in KLOTHO-deficient-mice. Recent evidence suggests that KLOTHO functions as a tumor suppressor by inhibiting Wnt signaling. KLOTHO gene silencing, including DNA methylation, has been observed in some human cancers. Aberrant activation of Wnt signaling plays a significant role in aging, and its silencing may be related to prostate cancer and other types of cancers. Thus, we investigated whether the expression of the anti-aging gene KLOTHO was associated with epigenetic changes in prostate cancer cell lines. KLOTHO mRNA was detected in the 22Rv1 cell line while it was not detected in DU145 and PC-3 cell lines. The restoration of KLOTHO mRNA in the DU145 and PC-3 cell lines was induced with a DNA methyltransferase inhibitor. Methylation-specific PCR was performed to determine the specific CpG sites in the KLOTHO promoter responsible for expression. In addition, the level of methylation was assessed in each CpG by performing bisulfite sequencing and quantitative pyrosequencing analysis. The results suggested a remarkable inverse relationship between KLOTHO expression and promoter methylation in prostate cancer cell lines.     

Quantitative DNA methylation analysis of genes coding for kallikrein-related peptidases 6 and 10 as biomarkers for prostate cancer

DNA methylation plays an important role in carcinogenesis and is being recognized as a promising diagnostic and prognostic biomarker for a variety of malignancies including Prostate cancer (PCa). The human kallikrein-related peptidases (KLKs) have emerged as an important family of cancer biomarkers, with KLK3, encoding for Prostate Specific Antigen, being most recognized. However, few studies have examined the epigenetic regulation of KLKs and its implications to PCa. To assess the biological effect of DNA methylation on KLK6 and KLK10 expression, we treated PC3 and 22RV1 PCa cells with a demethylating drug, 5-aza-2′deoxycytidine, and observed increased expression of both KLKs, establishing that DNA methylation plays a role in regulating gene expression. Subsequently, we have quantified KLK6 and KLK10 DNA methylation levels in two independent cohorts of PCa patients operated by radical prostatectomy between 2007–2011 (Cohort I, n = 150) and 1998–2001 (Cohort II, n = 124). In Cohort I, DNA methylation levels of both KLKs were significantly higher in cancerous tissue vs. normal. Further, we evaluated the relationship between DNA methylation and clinicopathological parameters. KLK6 DNA methylation was significantly associated with pathological stage only in Cohort I while KLK10 DNA methylation was significantly associated with pathological stage in both cohorts. In Cohort II, low KLK10 DNA methylation was associated with biochemical recurrence in univariate and multivariate analyses. A similar trend for KLK6 DNA methylation was observed. The results suggest that KLK6 and KLK10 DNA methylation distinguishes organ confined from locally invasive PCa and may have prognostic value.     

Quantitative DNA methylation analysis of genes coding for kallikrein-related peptidases 6 and 10 as biomarkers for prostate cancer

DNA methylation plays an important role in carcinogenesis and is being recognized as a promising diagnostic and prognostic biomarker for a variety of malignancies including Prostate cancer (PCa). The human kallikrein-related peptidases (KLKs) have emerged as an important family of cancer biomarkers, with KLK3, encoding for Prostate Specific Antigen, being most recognized. However, few studies have examined the epigenetic regulation of KLKs and its implications to PCa. To assess the biological effect of DNA methylation on KLK6 and KLK10 expression, we treated PC3 and 22RV1 PCa cells with a demethylating drug, 5-aza-2′deoxycytidine, and observed increased expression of both KLKs, establishing that DNA methylation plays a role in regulating gene expression. Subsequently, we have quantified KLK6 and KLK10 DNA methylation levels in two independent cohorts of PCa patients operated by radical prostatectomy between 2007–2011 (Cohort I, n = 150) and 1998–2001 (Cohort II, n = 124). In Cohort I, DNA methylation levels of both KLKs were significantly higher in cancerous tissue vs. normal. Further, we evaluated the relationship between DNA methylation and clinicopathological parameters. KLK6 DNA methylation was significantly associated with pathological stage only in Cohort I while KLK10 DNA methylation was significantly associated with pathological stage in both cohorts. In Cohort II, low KLK10 DNA methylation was associated with biochemical recurrence in univariate and multivariate analyses. A similar trend for KLK6 DNA methylation was observed. The results suggest that KLK6 and KLK10 DNA methylation distinguishes organ confined from locally invasive PCa and may have prognostic value.     

PCDH10 is required for the tumorigenicity of glioblastoma cells

Protocadherin10 (PCDH10)/OL-protocadherin is a cadherin-related transmembrane protein that has multiple roles in the brain, including facilitating specific cell–cell connections, cell migration and axon guidance. It has recently been reported that PCDH10 functions as a tumor suppressor and that its overexpression inhibits proliferation or invasion of multiple tumor cells. However, the function of PCDH10 in glioblastoma cells has not been elucidated. In contrast to previous reports on other tumors, we show here that suppression of the expression of PCDH10 by RNA interference (RNAi) induces the growth arrest and apoptosis of glioblastoma cells in vitro. Furthermore, we demonstrate that knockdown of PCDH10 inhibits the growth of glioblastoma cells xenografted into immunocompromised mice. These results suggest that PCDH10 is required for the proliferation and tumorigenicity of glioblastoma cells. We speculate that PCDH10 may be a promising target for the therapy of glioblastoma.     

Epigenetic changes in virus-associated human cancers

Epigenetics of human cancer becomes an area of emerging research direction due to a growing understanding of specific epigenetic pathways and rapid development of detection technologies. Aberrant promoter hypermethylation is a prevalent phenonmena in human cancers. Tumor suppressor genes are often hypermethylated due to the increased activity or deregulation of DNMTs. Increasing evidence also reveals that viral genes are one of the key players in regulating DNA methylation. In this review, we will focus on hypermethylation and tumor suppressor gene silencing and the signal pathways that are involved, particularly in cancers closely associated with the hepatitis B virus, simian virus 40 (SV40), and Epstein-Barr virus. In addition, we will discuss current technologies for genome-wide detection of epigenetically regulated targets, which allow for systematic DNA hypermethylation analysis. The study of epigenetic changes should provide a global view of gene profile in cancer, and epigenetic markers could be used for early detection, prognosis, and therapy of cancer.     

Differentially methylated genes and androgen receptor re-expression in small cell prostate carcinomas

Small cell prostate carcinoma (SCPC) morphology is rare at initial diagnosis but often emerges during prostate cancer progression and portends a dismal prognosis. It does not express androgen receptor (AR) or respond to hormonal therapies. Clinically applicable markers for its early detection and treatment with effective chemotherapy are needed. Our studies in patient tumor–derived xenografts (PDX) revealed that AR–negative SCPC (AR^?SCPC) expresses neural development genes instead of the prostate luminal epithelial genes characteristic of AR–positive castration-resistant adenocarcinomas (AR⁺ADENO). We hypothesized that the differences in cellular lineage programs are reflected in distinct epigenetic profiles. To address this hypothesis, we compared the DNA methylation profiles of AR^? and AR⁺ PDX using methylated CpG island amplification and microarray (MCAM) analysis and identified a set of differentially methylated promoters, validated in PDX and corresponding donor patient samples. We used the Illumina 450K platform to examine additional regions of the genome and the correlation between the DNA methylation profiles of the PDX and their corresponding patient tumors. Struck by the low frequency of AR promoter methylation in the AR^?SCPC, we investigated this region's specific histone modification patterns by chromatin immunoprecipitation. We found that the AR promoter was enriched in silencing histone modifications (H3K27me3 and H3K9me2) and that EZH2 inhibition with 3-deazaneplanocin A (DZNep) resulted in AR expression and growth inhibition in AR^?SCPC cell lines. We conclude that the epigenome of AR^? is distinct from that of AR⁺ castration-resistant prostate carcinomas, and that the AR^? phenotype can be reversed with epigenetic drugs.     

Epigenetic cancer therapy: Proof of concept and remaining challenges

Over the past few years several drugs that target epigenetic modifications have shown clinical benefits, thus seemingly validating epigenetic cancer therapy. More recently, however, it has become clear that these drugs are either characterized by low specificity or that their target enzymes have low substrate specificity. As such, clinical proof-of-concept for epigenetic cancer therapies remains to be established. Human cancers are characterized by widespread changes in their genomic DNA methylation and histone modification patterns. Epigenetic cancer therapy aims to restore normal epigenetic modification patterns through the inhibition of epigenetic modifier enzymes. In this review, we provide an overview about the known functional roles of DNA methyltransferases, histone deacetylases, histone methyltransferases, and demethylases in cancer development. The available data identify several examples that warrant further consideration as drug targets. Future research should be directed toward targeted enzyme inhibition and toward exploring interactions between epigenetic pathways to maximize cancer specificity.     

Epigenetic therapy approaches in non-small cell lung cancer: Update and perspectives

Non-small cell lung cancer (NSCLC) still constitutes the most common cancer-related cause of death worldwide. All efforts to introduce suitable treatment options using chemotherapeutics or targeted therapies have, up to this point, failed to exhibit a substantial effect on the 5-year-survival rate. The involvement of epigenetic alterations in the evolution of different cancers has led to the development of epigenetics-based therapies, mainly targeting DNA methyltransferases (DNMTs) and histone-modifying enzymes. So far, their greatest success stories have been registered in hematologic neoplasias. As the effects of epigenetic single agent treatment of solid tumors have been limited, the investigative focus now lies on combination therapies of epigenetically active agents with conventional chemotherapy, immunotherapy, or kinase inhibitors. This review includes a short overview of the most important preclinical approaches as well as an extensive discussion of clinical trials using epigenetic combination therapies in NSCLC, including ongoing trials. Thus, we are providing an overview of what lies ahead in the field of epigenetic combinatory therapies of NSCLC in the coming years.     

Epigenetic biomarkers in colorectal cancer: premises and prospects

Context: Colorectal cancer is one of the most common cancers worldwide. Epigenetic alterations play an important role in the pathogenesis of the colorectal cancer.

Objective: This review has focused on the most recent investigations, which has suggested potential epigenetic biomarkers in colorectal cancer.

Methods: Evidences were achieved by searching online medical databases including Google scholar, Pubmed, Scopus and Science Direct.

Results: Extensive studies have indicated that aberrant epigenetic modifications could serve as potential biomarkers for diagnosis, prognosis and prediction of colorectal cancer.

Conclusion: Advances in aberrant epigenetic modifications can open new avenues for exploration of reliable and robust biomarkers to improve the management of CRC patients.     

Multi-target siRNA based on DNMT3A/B homologous conserved region influences cell cycle and apoptosis of human prostate cancer cell line TSU-PR1

Abnormal genome hypermethylation participates in the tumorigenesis and development of prostate cancer. Prostate cancer cells highly express DNA methyltransferase 3 (DMNT3) family genes, essential for maintaining genome methylation. In the present study, multi-target siRNA, based on the homologous region of the DNMT3 family, was designed for the in vitro investigation of its effects on the proliferation, migration, and invasion of TSU-PR1 prostate cancer cells. The consequential cell-cycle derangement, through DNMT3A/B or only DNMT3B silencing, was partially efficient, without affecting apoptosis. DNMT3A silencing had absolutely no effect on changing TSU-PR1 cell biological behavior. Hence, DNMT3B alone apparently plays a key role in maintaining the unfavorable behavior of prostate-cancer cells, thereby implying its potential significance as a promising therapeutic target, with DNMT3A simply in the role of helper.     

Proteome analysis of human androgen-independent prostate cancer cell lines: variable metastatic potentials correlated with vimentin expression

To better understand the molecular mechanisms of prostate cancer (PCA) dissemination and to develop new anti-metastasis therapies, key regulatory molecules involved in PCA metastasis were identified in two human androgen-independent PCA cell lines, highly metastatic 1E8-H and lowly metastatic 2B4-L cells. Through 2-DE and MS analyses, 12 proteins with different expression levels in the two cell lines were identified. The following proteins were found to be significantly up-regulated in 1E8-H cells compared with 2B4-L cells: gp96 precursor, calreticulin precursor, vimentin (VIM), Hsp90alpha, peroxiredoxin 2, HNRPH1, ezrin, T-complex protein 1, alpha subunit, and hypothetical protein mln2339. In contrast, heart L-lactate dehydrogenase H chain, annexin I, and protein disulfide isomerase were notably down-regulated in 1E8-H cells compared with 2B4-L cells. To our knowledge, this study is the first to demonstrate that up-regulation of VIM expression positively correlates with the invasion and metastasis of androgen-independent PCA.     

EZH2, an epigenetic driver of prostate cancer

The histone methyltransferase EZH2 has been in the limelight of the fi eld of cancer epigenetics for a decade now since it was fi rst discovered to exhibit an elevated expression in metastatic prostate cancer. It persists to attract much scientifi c attention due to its important role in the process of cancer development and its potential of being an effective therapeutic target. Thus here we review the dysregulation of EZH2 in prostate cancer, its function, upstream regulators, downstream effectors, and current status of EZH2-targeting approaches. This review therefore provides a comprehensive overview of EZH2 in the context of prostate cancer.     

Death receptor-induced cell death in prostate cancer

Prostate cancer mortality results from metastasis and is often coupled with progression from androgen-dependent to androgen-independent growth. Unfortunately, no effective treatment for metastatic prostate cancer increasing patient survival is available. The absence of effective therapies reflects in part a lack of knowledge about the molecular mechanisms involved in the development and progression of this disease. Apoptosis, or programmed cell death, is a cell suicide mechanism that enables multicellular organisms to regulate cell number in tissues. Inhibition of apoptosis appears to be a critical pathophysiological factor contributing to the development and progression of prostate cancer. Understanding the mechanism(s) of apoptosis inhibition may be the basis for developing more effective therapeutic approaches. Our understanding of apoptosis in prostate cancer is relatively limited when compared to other malignancies, in particular, hematopoietic tumors. Thus, a clear need for a better understanding of apoptosis in this malignancy remains. In this review we have focused on what is known about apoptosis in prostate cancer and, more specifically, the receptor/ligand-mediated pathways of apoptosis as potential therapeutic targets.     

Genome-wide differentially methylated genes in prostate cancer tissues from African-American and Caucasian men

Increasing evidence suggests that aberrant DNA methylation changes may contribute to prostate cancer (PCa) ethnic disparity. To comprehensively identify DNA methylation alterations in PCa disparity, we used the Illumina 450K methylation platform to interrogate the methylation status of 485,577 CpG sites focusing on gene-associated regions of the human genome. Genomic DNA from African-American (AA; 7 normal and 3 cancers) and Caucasian (Cau; 8 normal and 3 cancers) was used in the analysis. Hierarchical clustering analysis identified probe-sets unique to AA and Cau samples, as well as common to both. We selected 25 promoter-associated novel CpG sites most differentially methylated by race (fold change > 1.5-fold; adjusted P < 0.05) and compared the β-value of these sites provided by the Illumina, Inc. array with quantitative methylation obtained by pyrosequencing in 7 prostate cell lines. We found very good concordance of the methylation levels between β-value and pyrosequencing. Gene expression analysis using qRT-PCR in a subset of 8 genes after treatment with 5-aza-2′-deoxycytidine and/or trichostatin showed up-regulation of gene expression in PCa cells. Quantitative analysis of 4 genes, SNRPN, SHANK2, MST1R, and ABCG5, in matched normal and PCa tissues derived from AA and Cau PCa patients demonstrated differential promoter methylation and concomitant differences in mRNA expression in prostate tissues from AA vs. Cau. Regression analysis in normal and PCa tissues as a function of race showed significantly higher methylation prevalence for SNRPN (P = 0.012), MST1R (P = 0.038), and ABCG5 (P < 0.0002) for AA vs. Cau samples. We selected the ABCG5 and SNRPN genes and verified their biological functions by Western blot analysis and siRNA gene knockout effects on cell proliferation and invasion in 4 PCa cell lines (2 AA and 2 Cau patients-derived lines). Knockdown of either ABCG5 or SNRPN resulted in a significant decrease in both invasion and proliferation in Cau PCa cell lines but we did not observe these remarkable loss-of-function effects in AA PCa cell lines. Our study demonstrates how differential genome-wide DNA methylation levels influence gene expression and biological functions in AA and Cau PCa.     

Prosaposin is a novel androgen-regulated gene in prostate cancer cell line LNCaP

Androgen-regulated genes (ARG) are implicated in normal and neoplastic growth of the prostate. Recently, we reported genomic amplification and/or overexpression of a previously known neurotrophic factor, prosaposin, in androgen-independent (AI) or metastatic prostate cancer (PCa) cells and tissues. Prosaposin and/or its known active molecular derivatives (e.g., saposin C) function as a pluripotent growth factor with diverse biological activities that favor malignant phenotypes in PCa cells. In addition, prosaposin or saposin C upregulates androgen receptor (AR) and AR-target genes (i.e., prostate-specific antigen, Probasin) expression and activity in LNCaP cells. Here, we examined prosaposin as an ARG. We report that DHT treatment of LNCaP cells increases prosaposin expression. In addition, we demonstrate androgen-responsiveness of prosaposin promoter and AR occupancy to a hormone-responsive element located in the proximal region of the prosaposin promoter. Our data for the first time identify prosaposin as an ARG. This observation, together with the pleiotropic growth factor activity of prosaposin, might suggest a role for this molecule in AR-dependent progression of prostate cancer at its early or late AI-state.